Prof. Manuel Espinosa Padrón (1942-2024): A Superb Plasmid Biologist and a Gentle Colossus.

Prof. Manuel Espinosa Padrón (1942-2024): A superb plasmid biologist and a gentle colossus.

The replication enhancer crtS depends on transcription factor Lrp for modulating binding of initiator RctB to ori2 of Vibrio cholerae.

Replication of Vibrio cholerae chromosome 2 (Chr2) initiates when the Chr1 locus, crtS (Chr2 replication triggering site) duplicates. The site binds the Chr2 initiator, RctB, and the binding increases when crtS is complexed with the transcription factor, Lrp. How Lrp increases the RctB binding and how RctB is subsequently activated for initiation by the crtS-Lrp complex remain unclear.

Kinetic principles of ParA2-ATP cycling guide dynamic subcellular localizations in Vibrio cholerae.

Dynamic protein gradients are exploited for the spatial organization and segregation of replicated chromosomes. However, mechanisms of protein gradient formation and how that spatially organizes chromosomes remain poorly understood. Here, we have determined the kinetic principles of subcellular localizations of ParA2 ATPase, an essential spatial regulator of chromosome 2 segregation in the multichromosome bacterium, Vibrio cholerae.

The linker domain of the initiator DnaA contributes to its ATP binding and membrane association in chromosomal replication.

DnaA, the initiator of chromosomal replication, has in its adenosine triphosphatase (ATPase) domain residues required for adenosine 5'-triphosphate (ATP) binding and membrane attachment. Here, we show that D118Q substitution in the DnaA linker domain, a domain known to be without major regulatory functions, influences ATP binding of DnaA and replication initiation in vivo. Although initiation defective by itself, overexpression of DnaA(D118Q) caused overinitiation of replication in ts cells and prevented cell growth.

The dimerization interface of initiator RctB governs chaperone and enhancer dependence of Vibrio cholerae chromosome 2 replication.

Protein function often requires remodeling of protein structure. In the well-studied iteron-containing plasmids, the initiator of replication has a dimerization interface that undergoes chaperone-mediated remodeling. This remodeling reduces dimerization and promotes DNA replication, since only monomers bind origin DNA.

sRNA-mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho-dependent transcription termination.

In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear.

Translation Initiation Control of RNase E-Mediated Decay of Polycistronic mRNA.

In bacteria, mRNA decay is a major mechanism for regulating gene expression. In , mRNA decay initiates with endonucleolytic cleavage by RNase E. Translating ribosomes impede RNase E cleavage, thus providing stability to mRNA.

Interactions of replication initiator RctB with single- and double-stranded DNA in origin opening of Vibrio cholerae chromosome 2.

Studies of bacterial chromosomes and plasmids indicate that their replication initiator proteins bind to origins of replication at many double-stranded sites and also at AT-rich regions where single-stranded DNA is exposed during origin opening. Single-strand binding apparently promotes origin opening by stabilizing an open structure, but how the initiator participates in this process and the contributions of the several binding sites remain unclear. Here, we show that the initiator protein of Vibrio cholerae specific to chromosome 2 (Chr2) also has single-strand binding activity in the AT-rich region of its origin.

A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution.

A Requirement for Global Transcription Factor Lrp in Licensing Replication of Chromosome 2.

The human pathogen, , belongs to the 10% of bacteria in which the genome is divided. Each of its two chromosomes, like bacterial chromosomes in general, replicates from a unique origin at fixed times in the cell cycle. Chr1 initiates first, and upon duplication of a site in Chr1, , Chr2 replication initiates.

Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage.

Initiation of chromosome replication in bacteria is precisely timed in the cell cycle. Bacteria that harbor multiple chromosomes face the additional challenge of orchestrating replication initiation of different chromosomes. In Vibrio cholerae, the smaller of its two chromosomes, Chr2, initiates replication after Chr1 such that both chromosomes terminate replication synchronously.

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Chromosome 2.

Replication of chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding.

Inactivation of Individual SeqA Binding Sites of the E. coli Origin Reveals Robustness of Replication Initiation Synchrony.

The Escherichia coli origin of replication, oriC, comprises mostly binding sites of two proteins: DnaA, a positive regulator, and SeqA, a negative regulator. SeqA, although not essential, is required for timely initiation, and during rapid growth, synchronous initiation from multiple origins. Unlike DnaA, details of SeqA binding to oriC are limited.

Random versus Cell Cycle-Regulated Replication Initiation in Bacteria: Insights from Studying Vibrio cholerae Chromosome 2.

Bacterial chromosomes initiate replication at a fixed time in the cell cycle, whereas there is generally no particular time for plasmid replication initiation or chromosomal replication initiation from integrated plasmids. In bacteria with divided genomes, the replication system of one of the chromosomes typically resembles that of bacteria with undivided genomes, whereas the remaining chromosomes have plasmid-like replication systems. For example, in Vibrio cholerae, a bacterium with two chromosomes (chromosome 1 [Chr1] and Chr2), the Chr1 system resembles that of the Escherichia coli chromosome, and the Chr2 system resembles that of iteron-based plasmids.

Opening the Strands of Replication Origins-Still an Open Question.

The local separation of duplex DNA strands (strand opening) is necessary for initiating basic transactions on DNA such as transcription, replication, and homologous recombination. Strand opening is commonly a stage at which these processes are regulated. Many different mechanisms are used to open the DNA duplex, the details of which are of great current interest.

Initiator protein dimerization plays a key role in replication control of Vibrio cholerae chromosome 2.

RctB, the initiator of replication of Vibrio cholerae chromosome 2 (chr2), binds to the origin of replication to specific 12-mer sites both as a monomer and a dimer. Binding to 12-mers is essential for initiation. The monomers also bind to a second kind of site, 39-mers, which inhibits initiation.

Chromosome segregation proteins of Vibrio cholerae as transcription regulators.

ABSTRACT Bacterial ParA and ParB proteins are best known for their contribution to plasmid and chromosome segregation, but they may also contribute to other cell functions. In segregation, ParA interacts with ParB, which binds to parS centromere-analogous sites. In transcription, plasmid Par proteins can serve as repressors by specifically binding to their own promoters and, additionally, in the case of ParB, by spreading from a parS site to nearby promoters.

Chromosome I controls chromosome II replication in Vibrio cholerae.

Control of chromosome replication involves a common set of regulators in eukaryotes, whereas bacteria with divided genomes use chromosome-specific regulators. How bacterial chromosomes might communicate for replication is not known. In Vibrio cholerae, which has two chromosomes (chrI and chrII), replication initiation is controlled by DnaA in chrI and by RctB in chrII.

Chromosome segregation in Vibrio cholerae.

The study of chromosome segregation is currently one of the most exciting research frontiers in cell biology. In this review, we discuss our current knowledge of the chromosome segregation process in Vibrio cholerae, based primarily on findings from fluorescence microscopy experiments. This bacterium is of special interest because of its eukaryotic feature of having a divided genome, a feature shared with 10% of known bacteria.

Evidence for two different regulatory mechanisms linking replication and segregation of vibrio cholerae chromosome II.

Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer).

Insensitivity of chromosome I and the cell cycle to blockage of replication and segregation of Vibrio cholerae chromosome II.

Unlabelled: Vibrio cholerae has two chromosomes (chrI and chrII) whose replication and segregation are under different genetic controls. The region covering the replication origin of chrI resembles that of the Escherichia coli chromosome, and both origins are under control of the highly conserved initiator, DnaA. The origin region of chrII resembles that of plasmids that have iterated initiator-binding sites (iterons) and is under control of the chrII-specific initiator, RctB.

Replication regulation of Vibrio cholerae chromosome II involves initiator binding to the origin both as monomer and as dimer.

The origin region of Vibrio cholerae chromosome II (chrII) resembles plasmid origins that have repeated initiator-binding sites (iterons). Iterons are essential for initiation as well as preventing over-initiation of plasmid replication. In chrII, iterons are also essential for initiation but over-initiation is prevented by sites called 39-mers.

Cell size and the initiation of DNA replication in bacteria.

In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ~30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation.

Chromosome dynamics in multichromosome bacteria.

On the basis of limited information, bacteria were once assumed to have no more than one chromosome. In the era of genomics, it has become clear that some, like eukaryotes, have more than one chromosome. Multichromosome bacteria provide opportunities to investigate how split genomes emerged, whether the individual chromosomes communicate to coordinate their replication and segregation, and what selective advantages split genomes might provide.