Biomedical subjects

Joe Pogliano

Publications and source records attributed to Joe Pogliano.

11 recordsLinked to original sources

PubMed2025

Disassembly activates Retron-Septu for antiphage defense.

Retrons are antiphage defense systems that produce multicopy single-stranded DNA (msDNA) and hold promise for genome engineering. However, the mechanisms of defense remain unclear. The Retron-Septu system integrates retron and Septu antiphage defenses. Cryo-electron microscopy structures reveal asymmetric nucleoprotein complexes comprising a reverse transcriptase, msDNA (a hybrid of msdDNA and msrRNA), and two PtuAB copies. msdDNA and msrRNA are essential for assembling this complex, with msrRNA adopting a conserved lariat-like structure that regulates reverse transcription. Notably, the assembled Retron-Septu complex is inactive, with msdDNA occupying the PtuA DNA binding site. Activation occurs upon disassembly, releasing PtuAB, which degrades single-stranded DNA to restrict phage replication. This "arrest-and-release" mechanism underscores the dynamic regulatory roles of msDNA, advancing our understanding of antiphage defense strategies.

Cryoelectron Microscopy↗

PubMed2024

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

Viral Proteins↗

PubMed2024

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

UNLABELLED: Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. SIGNIFICANCE STATEMENT: The phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.

Preprint↗

PubMed2006

DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development.

We here identify a protein (AlfA; actin like filament) that defines a new family of actins that are only distantly related to MreB and ParM. AlfA is required for segregation of Bacillus subtilis plasmid pBET131 (a mini pLS32-derivative) during growth and sporulation. A 3-kb DNA fragment encoding alfA and a downstream gene (alfB) is necessary and sufficient for plasmid stability. AlfA-GFP assembles dynamic cytoskeletal filaments that rapidly turn over (t(1/2)< approximately 45 s) in fluorescence recovery after photobleaching experiments. A point mutation (alfA D168A) that completely inhibits AlfA subunit exchange in vivo is strongly defective for plasmid segregation, demonstrating that dynamic polymerization of AlfA is necessary for function. During sporulation, plasmid segregation occurs before septation and independently of the DNA translocase SpoIIIE and the chromosomal Par proteins Soj and Spo0J. The absence of the RacA chromosome anchoring protein reduces the efficiency of plasmid segregation (by about two-fold), suggesting that it might contribute to anchoring the plasmid at the pole during sporulation. Our results suggest that the dynamic polymerization of AlfA mediates plasmid separation during both growth and sporulation.

Actin Cytoskeleton↗

PubMed2005

Bacterial DNA segregation by dynamic SopA polymers.

Many bacterial plasmids and chromosomes rely on ParA ATPases for proper positioning within the cell and for efficient segregation to daughter cells. Here we demonstrate that the F-plasmid-partitioning protein SopA polymerizes into filaments in an ATP-dependent manner in vitro, and that the filaments elongate at a rate that is similar to that of plasmid separation in vivo. We show that SopA is a dynamic protein within the cell, undergoing cycles of polymerization and depolymerization, and shuttling back and forth between nucleoprotein complexes that are composed of the SopB protein bound to sopC-containing plasmids (SopB/sopC). The dynamic behavior of SopA is critical for Sop-mediated plasmid DNA segregation; mutations that lock SopA into a static polymer in the cell inhibit plasmid segregation. We show that SopA colocalizes with SopB/sopC in the cell and that SopB/sopC nucleates the assembly of SopA and is required for its dynamic behavior. When SopA is polymerized in vitro in the presence of SopB and sopC-containing DNA, SopA filaments emanate from the plasmid DNA in radial asters. We propose a mechanism in which plasmid separation is driven by the polymerization of SopA, and we speculate that the radial assembly of SopA polymers is responsible for positioning plasmids both before and after segregation.

Bacterial Proteins↗

PubMed2004

DNA segregation by bacterial actin homologs.

The bacterial actin homolog ParM catalyzes segregation of plasmid DNA in E. coli. Recent studies now suggest a model in which ParM forms actin-like filaments between two plasmid molecules, thereby providing the driving force for plasmid DNA separation.

Actin Cytoskeleton↗

PubMed2003

Chromosome segregation in Eubacteria.

It is now clear that bacterial chromosomes rapidly separate in a manner independent of cell elongation, suggesting the existence of a mitotic apparatus in bacteria. Recent studies of bacterial cells reveal filamentous structures similar to the eukaryotic cytoskeleton, proteins that mediate polar chromosome anchoring during Bacillus subtilis sporulation, and SMC interacting proteins that are involved in chromosome condensation. A picture is thereby developing of how bacterial chromosomes are organized within the cell, how they are separated following duplication, and how these processes are coordinated with the cell cycle.

Bacteria↗

PubMed2002

Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli.

Targeting of DNA molecules to specific subcellular positions is essential for efficient segregation, but the mechanisms underlying these processes are poorly understood. In Escherichia coli, several plasmids belonging to different incompatibility groups (F, P1 and RK2) localize preferentially near the midcell and quartercell positions. Here we compare the relative positions of these three plasmids using fluorescence in situ hybridization. When plasmids F and P1 were localized simultaneously using differentially labeled probes, the majority of foci (approximately 75%) were well separated from each other. Similar results were found when we compared the subcellular localization of F with RK2, and RK2 with P1: regardless of the number of foci per cell or growth conditions, most of the foci (70-80%) were not in close proximity to one another. We also localized RK2 in Pseudomonas aeruginosa and Vibrio cholerae, and found that plasmid RK2 localization is conserved across bacterial species. Our results suggest that each plasmid has its own unique subcellular address, implying a mechanism for the stable co-existence of plasmids in which subcellular targeting plays a major role.

Cell Division↗

PubMed2002

Dynamic cellular location of bacterial plasmids.

Recent studies have shown that plasmids are organized inside bacterial cells in a remarkably complex way. Plasmids containing active partitioning systems are tethered to specific regions of the cell, and the number and position of plasmid molecules within the cell are coordinated with the bacterial host cell cycle and growth rate. Plasmids belonging to different incompatibility groups are also tethered to different sites within the cell, and segregate at different times relative to one another and to the bacterial cell cycle. Recent studies suggest that many of these observations regarding subcellular plasmid dynamics formulated for Escherichia coli plasmids may be broadly conserved.

Bacterial Proteins↗

PubMed2002

ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase.

Broad-host-range plasmid RK2 encodes a post-segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication-proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase- and gyrase-dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase-DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.

Adenosine Triphosphate↗

PubMed2002

Partitioning of chromosomal DNA during establishment of cellular asymmetry in Bacillus subtilis.

The switch from symmetric to asymmetric cell division is a key feature of development in many organisms, including Bacillus subtilis sporulation. Here we demonstrate that, prior to the onset of asymmetric cell division, the B. subtilis chromosome is partitioned into two unequally sized domains, with the origin-proximal one-third of the future forespore chromosome condensed near one pole of the cell. Asymmetric chromosome partitioning is independent of polar division, as it occurs in cells depleted of FtsZ but depends on two transcription factors that govern the initiation of sporulation, sigma(H) and Spo0A-P. It is also independent of chromosome partitioning proteins Spo0J and Soj, suggesting the existence of a novel mechanism controlling chromosome structure. Thus, our results demonstrate that, during sporulation, two separable events prepare B. subtilis for asymmetric cell division: the relocation of cell division sites to the cell poles and the asymmetric partitioning of the future forespore chromosome.

Bacillus subtilis↗