Considerations on bacterial nucleoids

The classic genome organization of the bacterial chromosome is normally envisaged with all its genetic markers linked, thus forming a closed genetic circle of duplex stranded DNA (dsDNA) and several proteins in what it is called as “the bacterial nucleoid.” This structure may be more or less corrugated depending on the physiological state of the bacterium (i.e., resting state or active growth) and is not surrounded by a double membrane as in eukayotic cells. The universality of the closed circle model in bacteria is however slowly changing, as new data emerge in different bacterial groups such as in Planctomycetes and related microorganisms, species of Borrelia, Streptomyces, Agrobacterium, or Phytoplasma. In these and possibly other microorganisms, the existence of complex formations of intracellular membranes or linear chromosomes is typical; all of these situations contributing to weakening the current cellular organization paradigm, i.e., prokaryotic vs eukaryotic cells.     

Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii~1

Dinoflagellate is one of the primitive eukaryotes,whosenucleus may represent one of the transition stages fromprokaryotic nucleoid to typical eukaryotic nucleus.Usingselective extraction together with embeddment-free sectionand whole mount electron microscopy,a delicate nuclearmatrix filament network was shown,for the first time,indinoflagellate Crypthecodinium cohnii nucleus.Chromosomeresidues are connected with nuclear matrix filaments to forma complete network spreading over the nucleus.Moreover,we demonstrated that the dinoflagellate chromosome retainsa protein scaffold after the depletion of DNA and solubleproteins.This scaffold preserves the characteristic mor-phology of the chromosome.Two dimensional elec-trophoreses indicated that the nuclear matrix and chromo- some scaffold are mainly composed of acidic proteins.Ourresults demonstrated that a framework similar to the nuclearmatrix and chromosome scaffold in mammalian cells appearsin this primitive eukaryote,suggesting that these structuresmay have been originated from the early stages of eukaryoteevolution.     

The architectural role of nucleoid-associated proteins in the organization of bacterial chromatin: a molecular perspective

The bacterial genome is folded into a compact structure called the nucleoid. Considerable compaction of the DNA molecule is required in order to reduce its volume below that of the cell. Several mechanisms, such as molecular crowding and DNA supercoiling contribute to the compactness of the nucleoid. Besides these mechanisms, a number of architectural proteins associate with the chromosomal DNA and cause it to fold into a compact structure by bridging, bending or wrapping DNA. In this review, we provide an overview of the major nucleoid-associated proteins from a structural perspective and we discuss their possible roles in dynamically shaping the bacterial nucleoid.     

Unfolding of the bacterial nucleoid both in vivo and in vitro as a result of exposure to camphor.

Both prokaryotic and eukaryotic cells are sensitive to killing by camphor; however, the mechanism by which camphor kills has not been elucidated. We report here that camphor unfolds the nucleoid of Escherichia coli and that unfolding does not require DNA replication, translation, or cell division. We show that exposure of isolated nucleoids to camphor results in unfolding of the chromosome.     

New insights in the ϕ29 terminal protein DNA‐binding and host nucleoid localization functions

Protein‐primed DNA replication constitutes a strategy to initiate viral DNA synthesis in a variety of prokaryotic and eukaryotic organisms. Although the main function of viral terminal proteins (TPs) is to provide a free hydroxyl group to start initiation of DNA replication, there are compelling evidences that TPs can also play other biological roles. In the case of Bacillus subtilis bacteriophage ?29, the N‐terminal domain of the TP organizes viral DNA replication at the bacterial nucleoid being essential for an efficient phage DNA replication, and it contains a nuclear localization signal (NLS) that is functional in eukaryotes. Here we provide information about the structural properties of the ?29 TP N‐terminal domain, which possesses sequence‐independent DNA‐binding capacity, and dissect the amino acid residues important for its biological function. By mutating all the basic residues of the TP N‐terminal domain we identify the amino acids responsible for its interaction with the B. subtilis genome, establishing a correlation between the capacity of DNA‐binding and nucleoid localization of the protein. Significantly, these residues are important to recruit the DNA polymerase at the bacterial nucleoid and, subsequently, for an efficient phage DNA replication.     

Interactions stabilizing DNA tertiary structure in the Escherichia coli chromosome investigated with ionizing radiation

The structure of the bacterial chromosome was investigated after introducing breaks in the DNA with gamma irradiation. It is demonstrated that irradiation of the chromosome in the cell prior to isolation results in partial unfolding of the isolated condensed DNA, while irradiation of the chromosome after it is released from the cell has no demonstrable effect on DNA folding. The results indicate that RNA/DNA interactions which stabilize DNA folds are unstable when breaks are introduced in the DNA prior to isolation of the chromosome. It is suggested that the supercoiled state of the DNA is required for the initial stabilization of some of the critical RNA/DNA interaction in the isolated nucleoid. However, some of these interactions are not affected by irradiation of the cells. Remnant supercoiling in partially relaxed chromosomes containing a limited number of DNA breaks has the same superhelical density as the unirradiated chromosome. This suggests that restraints on rotation of the packaged DNA are formed prior to the physical unwinding which occurs at the sites of the radiation induced DNA breaks. — Analysis of the in vitro irradiated chromosomes shows that there are 100+-30 domains of supercoiling per genome equivalent of DNA. The introduction of up to 50 double-strand breaks per nucleoid does not influence rotor speed effects of the sedimentation coefficient of the chromosome.     

The bacterial nucleoid visualized by fluorescence microscopy of cells lysed within agarose: comparison of Escherichia coli and spirochetes of the genus Borrelia.

The nucleoids of Escherichia coli and the spirochetes Borrelia burgdorferi and Borrelia hermsii, agents of Lyme disease and relapsing fever, were examined by epifluorescence microscopy of bacterial cells embedded in agarose and lysed in situ with detergent and protease. The typical E. coli nucleoid was a rosette in which 20 to 50 long loops of DNA emanated from a dense node of DNA. The percentages of cells in a population having nucleoids with zero, one, two, and three nodes varied with growth rate and growth phase. The borrelia nucleoid, in contrast, was a loose network of DNA strands devoid of nodes. This nucleoid structure difference correlates with the unusual genome of Borrelia species, which consists primarily of linear replicons, including a 950-kb linear chromosome and linear plasmids. This method provides a simple, direct means to analyze the structure of the bacterial nucleoid.     

On a possible relationship between bacterial endospore formation and the origin of eukaryotic cells

The acquisition of intracellular organelles, including mitochondria and plastids and a membrane-bounded nucleus, have been postulated to be key events in the development of the eukaryotic from the prokaryotic ancestral cell. The two major hypotheses to account for such acquisitions are: (1) primitive cells originally obtained organelles by engulfing free-living prokaryotes which then entered into symbiotic association (“endosymbiosis”) with them; (2) organelles arose through the engulfment by the primitive cell of part of its own cytoplasm. To some extent, the former hypothesis has received most support, because endosymbiosis is known to occur in extant organisms, whilst the latter hypothesis has received less support, because cytoplasmic engulfment by prokaryotes is not now thought to occur. However, during the process of endospore formation by extant bacteria, the protoplast within the single cell is observed to divide in a unique manner such that the cell in effect engulfs a portion of its own cytoplasm. The process is strikingly similar to the engulfment suggested by the second hypothesis to have initiated the evolution of eukaryotes. The engulfed cytoplasm is bounded by a double membrane within the “mother cell” and contains enzymes, ribosomes and a complete genome. In many respects this parallels the supposed primitive eukaryotic state and, it is argued, confers potential advantages on the cell, particularly through the control that the “mother cell” can exert on the enclosed compartment. It is hypothesized that bacterial endospore formation is therefore one product of evolution from an early engulfment event that led also to the development of complex eukaryotic cells.     

The bacterial nucleoid: a highly organized and dynamic structure

Recent advances in bacterial cell biology have revealed unanticipated structural and functional complexity, reminiscent of eukaryotic cells. Particular progress has been made in understanding the structure, replication, and segregation of the bacterial chromosome. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. The position of genetic loci on the chromosome is thereby linearly correlated with their position in the cell. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Moreover, active transport processes ensure the efficient segregation of sister chromosomes and the faithful restoration of nucleoid organization while DNA replication and condensation are in progress.     

Formation of a micronuclei-like structure induced by colchicine treatment in Saccharomyces cerevisiae

Minute nuclei named “smaller nuclei” were generated when the cells of Saccharomyces cerevisiae were treated with colchicine. The formation of “smaller nuclei” seemed to be related to nuclear division because those nuclei were only produced under conditions suitable for nuclear division. The fact that the average DNA content of “smaller nuclei” was almost one tenth of that of the isolated normal diploid nuclei showed that the “smaller nuclei” are not condensed nuclei but aneuploid nuclei like micronuclei in animal cells. It appeared therefore likely that a micronuclei-like structure could be produced by colchicine treatment in S. cerevisiae.     

Was the evolution of plastid genetic machinery discontinuous?

The plastid nucleoid consists of plastid DNA and various, mostly uncharacterized, DNA-binding proteins. The plastid DNA undoubtedly originated from an ancestral cyanobacterial genome, but the origin of the nucleoid proteins appears complex. Initial biochemical analysis of these proteins, as well as comparative genome informatics, suggest that proteins of eukaryotic origin replaced most of the original prokaryotic proteins during the evolution of plastids in the lineage of green plants.     

Induction of chromosome damage by restriction enzymes during mitosis.

Once electroporated into the nucleus of eukaryotic cells, restriction enzymes will bind at specific DNA sequences and cleave DNA to make double-strand breaks. These induced breaks can lead to chromosome aberrations and consequently offer one approach to determining the mechanism(s) of aberration formation. Because the higher-order structure of DNA in eukaryotic cells might influence the ability of restriction enzymes to locate their recognition sequence, bind, and cleave DNA, we have investigated whether enzymes will cut DNA during metaphase when the chromosomes are most condensed. Chinese hamster ovary cells synchronized in mitosis and treated with either AluI or Sau3AI showed few chromosome aberrations when held in mitosis for 1, 2, or 3 h after enzyme treatment. However, some disruption of chromosome morphology was seen, especially after exposure to Sau3AI. When cells were allowed to complete one cell cycle after enzyme treatment in the preceding mitosis, there was extensive chromosome damage, with the most abundant type of lesion being the interstitial deletion. It appears that restriction enzymes will cleave the highly condensed DNA in mitotic cells but that decondensation, DNA replication, and recondensation are required before the aberrations are manifested.