Biomedical subjects

G T Marczynski

Publications and source records attributed to G T Marczynski.

At least 19 recordsLinked to original sources

PubMed2001

Replication intermediate analysis confirms that chromosomal replication origin initiates from an unusual intergenic region in Caulobacter crescentus.

The alpha-proteobacterium Caulobacter crescentus possesses a developmental cell cycle that restricts chromosome replication to a stalked cell type. The proposed C.crescentus chromosome replication origin (Cori) lies between hemE and RP001, an unusual intergenic region not previously associated with bacterial replication origins, although a similar genomic arrangement is also present at the putative replication origin in the related bacterium Rickettsia prowazekii. The cloned Cori supports autonomous plasmid replication selectively in the stalked cell type implying that replication of the entire chromosome also initiates between hemE and RP001. To confirm this location, we applied the 2-D (N/N) agarose gel electrophoresis technique to resolve and identify chromosome replication intermediates throughout a 30 kb region spanning Cori. Replication initiation in Cori was uniquely characterized by an 'origin bubble and Y-arc' pattern and this observation was supported by simple replication fork 'Y-arc' patterns that characterized the regions flanking Cori. These replication forks originated bi-directionally from within Cori as determined by the fork direction assay. Therefore, chromosomal replication initiates from the unusual hemE/RP001 intergenic region that we propose represents a new class of replication origins.

Blotting, Southern↗

PubMed2001

Physiological consequences of blocked Caulobacter crescentus dnaA expression, an essential DNA replication gene.

Caulobacter crescentus chromosome replication is precisely coupled to a developmental cell cycle. Like most eubacteria, C. crescentus has a DnaA homologue that is presumed to initiate chromosome replication. However, the C. crescentus replication origin (Cori) lacks perfect consensus Escherichia coli DnaA boxes. Instead, the Cori strong transcription promoter (Ps) may regulate chromosome replication through the CtrA cell cycle response regulator. We therefore created a conditional dnaA C. crescentus strain. Blocking dnaA expression immediately decreased DNA synthesis, which stopped after approximately one doubling period. Fluorescent flow cytometry confirmed that DNA synthesis is blocked at the initiation stage. Cell division also stopped, but not swarmer to stalked cell differentiation. All cells became stalked cells that grew as long filaments. Therefore, general transcription and protein synthesis continued, whereas DNA synthesis stopped. However, transcription was selectively blocked from the flagellar fliQ and fliL and methyltransferase ccrM promoters, which require CtrA and are blocked by different DNA synthesis inhibitors. Interestingly, transcription from Cori Ps continued unaltered. Therefore, Ps transcription is not sufficient for chromosome replication. Approximately 6-8 h after blocked dnaA expression, cells lost viability exponentially. Coincidentally, beta-galactosidase was induced from one transcription reporter, suggesting an altered physiology. We conclude that C. crescentus DnaA is essential for chromosome replication initiation, and perhaps also has a wider role in cell homeostasis.

Bacterial Proteins↗

PubMed2001

Conserved gene cluster at replication origins of the alpha-proteobacteria Caulobacter crescentus and Rickettsia prowazekii.

A 30-kb region surrounding the replication origin in Caulobacter crescentus was analyzed. Comparison to the genome sequence of another alpha-proteobacterium, Rickettsia prowazekii, revealed a conserved cluster of genes (RP001, hemE, hemH, and RP883) that overlaps the established origin of replication in C. crescentus and the putative origin of replication in R. prowazekii. The genes flanking this cluster differ between these two organisms. We therefore propose that this conserved gene cluster can be used to identify the origin of replication in other alpha-proteobacteria.

Alphaproteobacteria↗

PubMed2000

Analysis of a cell-cycle promoter bound by a response regulator.

Caulobacter crescentus CtrA protein, an OmpR-type response regulator, receives cell-cycle signals and binds the proposed consensus TTAA-N7-TTAA present inside the chromosome replication origin and cell-cycle transcription promoters. We synthesized a 42 bp cell-cycle promoter based on this consensus and elements of the fliL promoter. Over 100 promoter mutations were assayed for transcription directed by CtrA. Although both CtrA binding half-sites cooperate and the N7 spacing is critical for transcription, the upstream half-site is relatively flexible. The downstream CtrA half-site is less flexible and more important for cell-cycle regulation. A CtrA binding site and a -10 promoter element are sufficient for cell-cycle transcription, and both sequences cooperate and compensate for respective defects. Mutations in the CtrA binding site, but not in the -10 promoter sequence, perturb cell-cycle transcription. A single base-pair change switches a cell-cycle promoter into a strong conventional promoter. We propose rules for CtrA binding and promoter interactions implying how CtrA evolved into a versatile regulator of cell-cycle functions including flagellar biogenesis, cell division, DNA methylation, and chromosome replication.

Bacterial Proteins↗

PubMed2000

Cell cycle regulator phosphorylation stimulates two distinct modes of binding at a chromosome replication origin.

In Caulobacter crescentus, the global response regulator CtrA controls chromosome replication and determines the fate of two different cell progenies. Previous studies proposed that CtrA represses replication by binding to five sites, designated [a-e], in the replication origin. We show that phosphorylated CtrA binds sites [a-e] with 35- to 100-fold lower K(d) values than unphosphorylated CtrA. CtrA phosphorylation stimulates two distinct modes of binding to the replication origin. Phosphorylation stimulates weak intrinsic protein-protein cooperation between half-sites and does not stimulate CtrA-P binding unless protein-DNA contacts are made at both half-sites. CtrA phosphorylation also stimulates cooperative binding between complete sites [a] and [b]. However, binding to each of the other CtrA-binding sites [c], [d] and [e] is completely independent and suggests a modular organization of replication control by CtrA. We therefore propose a model where a phosphorelay targets separate biochemical activities inside the replication origin through both cooperative and independent CtrA-binding sites.

Bacterial Proteins↗

PubMed2000

Selective cell cycle transcription requires membrane synthesis in Caulobacter.

Caulobacter crescentus divides asymmetrically and creates distinct polar membrane surfaces that partition during the cell cycle to distinct cell progeny. Blocking membrane synthesis prevented transcription from selective promoters involved in asymmetric cell division. Transcription from sigma-54-dependent flagellar promoters was blocked completely; however, transcription from the CtrA response regulator-dependent flagellar promoters was activated but reduced. Transcription from the ccrM (DNA methylation) promoter and the che (chemosensory) promoter was also blocked completely. Transcription from a strong promoter at the chromosome replication origin was first stopped then induced by blocked membrane synthesis. We propose a feedback control coupling membrane synthesis to transcription that selectively supports membrane-associated processes such as flagellar assembly, chemosensory biogenesis and chromosome replication.

Caulobacter crescentus↗

PubMed2000

Transcription reporters that shuttle cloned DNA between high-copy Escherichia coli plasmids and low-copy broad-host-range plasmids.

We describe and apply lacZ transcription reporter plasmids designed for both biochemical analyses requiring high DNA yield and physiological studies requiring low gene dosage. Standard DNA ligations are performed at seven unique restriction sites 5' to the lacZ gene on high-copy ColE1 plasmids suitable for double- or single-strand DNA sequencing. A divergent gusA transcription reporter is included and serves as an internal control. Rec(+) Escherichia coli cells readily shuttle DNA placed between gusA and lacZ by allelic exchange with pRK290-based plasmids that subsequently conjugate and replicate in most gram-negative bacteria. We applied this system to study Caulobacter crescentus cell cycle promoters directed by the CtrA response-regulator protein. Synthetic oligonucleotides were ligated to create altered CtrA binding sites and corresponding promoters with varied transcription strength. We also document the phenomenon of long-range promoter interference. A strong promoter can repress up to twofold the transcription from a divergent promoter located 100 bp away. However, the cell cycle timing of both promoters is not changed. Additional applications of our system and theoretical aspects of promoter organization are discussed.

Bacterial Proteins↗

PubMed1999

Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus.

Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.

Caulobacter crescentus↗

PubMed1998

Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin.

Caulobacter crescentus divides asymmetrically generating two distinct cell types at each cell division: a stalked cell competent for DNA replication, and a swarmer cell that is unable to initiate DNA replication until it differentiates into a stalked cell later in the cell cycle. The CtrA protein, a member of the response regulator family of the two-component signal transduction system, controls multiple cell cycle processes in Caulobacter and is present in swarmer cells but absent from stalked cells. We report that CtrA binds five sites within the chromosome replication origin in vitro. These sites overlap an essential DnaA box and a promoter in the origin that is essential for replication initiation. Analysis of mutant alleles of ctrA and point mutations in one of the CtrA binding sites in the origin demonstrate that CtrA represses replication in vivo. CtrA-mediated repression at the origin thus restricts replication to the stalked cell type. Thus, the direct coupling of chromosome replication with the cell cycle is mediated by the ubiquitous two-component signaling proteins.

Alleles↗

PubMed1996

Cell cycle control by an essential bacterial two-component signal transduction protein.

Dividing cells must coordinate cell cycle events to ensure genetic stability. Here we identify an essential two-component signal transduction protein that controls multiple events in the Caulobacter cell cycle, including cell division, stalk synthesis, and cell cycle-specific transcription. This protein, CtrA, is homologous to response regulator transcription factors and controls transcription from a group of cell cycle-regulated promoters critical for DNA replication, DNA methylation, and flagellar biogenesis. CtrA activity in the cell cycle is controlled both transcriptionally and by phosphorylation. As purified CtrA binds an essential DNA sequence motif found within its target promoters, we propose that CtrA acts in a phosphorelay signal transduction system to control bacterial cell cycle events directly at the transcriptional level.

Amino Acid Sequence↗

PubMed1995

A developmentally regulated chromosomal origin of replication uses essential transcription elements.

Only one of the two chromosomes in the asymmetric Caulobacter predivisional cell initiates replication in the progeny cells. Transcription from a strong promoter within the origin occurs uniquely from the replication-competent chromosome at the stalked pole of the predivisional cell. This regulated promoter has an unusual sequence organization, and transcription from this promoter is essential for regulated (cell type-specific) replication. Our analysis defines a new class of bacterial origins and suggests a coupling between transcription and replication that is consistent with the phylogenetic relationship of Caulobacter to the ancestral mitochondrion.

Base Sequence↗

PubMed1995

The control of asymmetric gene expression during Caulobacter cell differentiation.

The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. Each cell division produces two distinct cell types: a swarmer cell and a stalked cell. These cells possess distinct functional morphologies and differential programs of transcription and DNA replication. The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry. The molecular details of these universal cellular processes in C. crescentus will provide paradigms applicable to many general aspects of cellular differentiation.

Bacterial Proteins↗

PubMed1993

Gal4 protein binding is required but not sufficient for derepression and induction of GAL2 expression.

The Saccharomyces cerevisiae GAL2 gene upstream activator sequence (UAS) region was examined for protein bound in vivo by chromatin footprinting at high resolution. Gal4 transcriptional activator protein binds to the two consensus UAS sites whether GAL2 expression is induced, uninduced, or repressed by growth with different carbon sources. Although wild type strains show loss of the Gal4 protein-specific footprint in repressing media containing glucose, constitutive high level expression of Gal4 protein restores the GAL2 UAS footprints without fully derepressing GAL2 transcription. Thus binding of the Gal4 activator to target sites in the DNA is required but not sufficient for GAL2 derepression and induction. Gal4-independent protein-DNA complexes were also detected in the region, including one over the previously noted centromere-binding protein (CP1) site upstream of the Gal4 complexes.

Base Sequence↗

PubMed1993

Bacterial chromosome origins of replication.

Bacteria regulate chromosomal replication from one specific origin. We compare the regulatory requirements, DNA structures, and biochemical properties of the prototypic Escherichia coli origin with those of evolutionarily distant Bacillus subtilis and Caulobacter crescentus origins. The ubiquitous DnaA protein is a major regulator of all three bacterial origins. Unique features of these origins, however, may reflect specific regulatory requirements placed on them.

Bacteria↗

PubMed1992

Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus.

Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur. In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. The C. crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. This implies that cis-acting replication control elements are closely linked to this origin of replication. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. This origin also possesses three additional motifs that are unique to the C. crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication.

Bacterial Proteins↗

PubMed1990

Plasmid and chromosomal DNA replication and partitioning during the Caulobacter crescentus cell cycle.

Cell division in Caulobacter crescentus yields a swarmer and a stalked cell. Only the stalked cell progeny is able to replicate its chromosome, and the swarmer cell progeny must differentiate into a stalked cell before it too can replicate its chromosome. In an effort to understand the mechanisms that limit chromosomal replication to the stalked cell, plasmid DNA synthesis was analyzed during the developmental cell cycle of C. crescentus, and the partitioning of both the plasmids and the chromosomes to the progeny cells was examined. Unlike the chromosome, plasmids from the incompatibility groups Q and P replicated in all C. crescentus cell types. However, all plasmids tested showed a ten- to 20-fold higher replication rate in the stalked cells than the swarmer cells. We observed that all plasmids replicated during the C. crescentus cell cycle with comparable kinetics of DNA synthesis, even though we tested plasmids that encode very different known (and putative) replication proteins. We determined the plasmid copy number in both progeny cell types, and determined that plasmids partitioned equally to the stalked and swarmer cells. We also reexamined chromosome partitioning in a recombination-deficient strain of C. crescentus, and confirmed an earlier report that chromosomes partition to the progeny stalked and swarmer cells in a random manner that does not discriminate between old and new DNA strands.

Cell Cycle↗

PubMed1990

Regulatory interactions between phospholipid synthesis and DNA replication in Caulobacter crescentus.

Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes. A C. crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. The first effect of withholding supplement was the cessation of synthesis of phosphatidylglycerol, a major component of the C. crescentus membrane. In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. However, after replication proceeded bidirectionally for a short time, DNA synthesis dropped to a low level. The cell cycle blocked at a distinct middivision stalked cell, and this was followed by cell death. The "glycerol-less" death of the gpsA mutant could be prevented if the cells were treated with novobiocin to prevent the initiation of DNA replication. Our observations suggest that the processivity of C. crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome.

Bacteria↗

PubMed1989

Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro.

We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested.

Base Sequence↗