Ndd, the Bacteriophage T4 Protein That Disrupts the Escherichia coli Nucleoid, Has a DNA Binding Activity

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.     

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp. Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the C31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of C31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.     

Variation of the folding and dynamics of the Escherichia coli chromosome with growth conditions

We examine whether the Escherichia coli chromosome is folded into a self‐adherent nucleoprotein complex, or alternately is a confined but otherwise unconstrained self‐avoiding polymer. We address this through in vivo visualization, using an inducible GFP fusion to the nucleoid‐associated protein Fis to non‐specifically decorate the entire chromosome. For a range of different growth conditions, the chromosome is a compact structure that does not fill the volume of the cell, and which moves from the new pole to the cell centre. During rapid growth, chromosome segregation occurs well before cell division, with daughter chromosomes coupled by a thin inter‐daughter filament before complete segregation, whereas during slow growth chromosomes stay adjacent until cell division occurs. Image correlation analysis indicates that sub‐nucleoid structure is stable on a 1 min timescale, comparable to the timescale for redistribution time measured for GFP–Fis after photobleaching. Optical deconvolution and writhe calculation analysis indicate that the nucleoid has a large‐scale coiled organization rather than being an amorphous mass. Our observations are consistent with the chromosome having a self‐adherent filament organization.     

Association of R6K plasmid replicative intermediates with the folded chromosome of Escherichiacoli

When $\text{Escherichia}\text{coli}$ strain CSH50(R6K) is lysed so as to preserve the folded chromosome structure approximately 9 of the 11 R6K molecules maintained per chromosomal equivalent cosediment with the host nucleoid on a neutral sucrose gradient; the remaining 2 plasmids sediment at their normal rate. When cells are briefly labeled with [³H]thymidine, the majority of plasmid replicative intermediates and nascent mature plasmids are found in the plasmid subpopulation that cosediments with host folded chromosomes. This finding suggests that plasmid replication occurs in a restricted cellular locus, perhaps even while in association with its host's folded chromosome.     

Integration Host Factor of Mycobacterium tuberculosis,mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.     

E. coli Fis Protein Insulates the cbpA Gene from Uncontrolled Transcription

The Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and co-chaperone. It is expressed at high levels as cells enter stationary phase. Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. We show that Fis, the dominant growth-phase nucleoid protein, prevents CbpA expression in growing cells. Regulation by Fis involves an unusual “insulation” mechanism. Thus, Fis protects cbpA from the effects of a distal promoter, located in an adjacent gene. In stationary phase, when Fis levels are low, CbpA binds the E. coli chromosome with a preference for the intrinsically curved Ter macrodomain. Disruption of the cbpA gene prompts dramatic changes in DNA topology. Thus, our work identifies a novel role for Fis and incorporates CbpA into the growing network of factors that mediate bacterial chromosome structure.     

Application of Physical Methods to the Study of Serum Lipoprotein

Abstract

One of the primary characteristics distinguishing prokaryotic from eukaryotic cells is the absence of a nucleus with a clearly defined nuclear membrane. In prokaryotic cells the DNA is condensed into a structure called the nucleoid. This structure has also been referred to attimes as the nuclear body, prokaryotic nucleus, bacterial chromosome, folded genome, or folded bacterial chromosome. The nomenclature sometimes becomes confusing because unfolded bacterial DNA free of other components of the nucleoid has also been referred to as the bacterial chromosome. To avoid such confusion, it would be preferable to reserve the terms nucleoid or bacterial chromosome to describe the condensed prokaryotic DNA structures which have some features analogous to the eukaryotic metaphase chromosome and condensed interphase chromatin. If this convention is followed, the terms “folded chromosome” or “folded genome” become ambiguous because they could equally mean “folded nucleoid.” These latter terms will, therefore, be avoided throughout this article.     

Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA

HU (Histone‐like protein from Escherichia coli strain U93) is the most conserved nucleoid‐associated protein in eubacteria, but how it impacts global chromosome organization is poorly understood. Using single‐molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface‐exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUα(triKA) mutant results in an over‐condensed and mis‐segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUα subunit, deleting the HUβ subunit, or deleting nucleoid‐associated naRNAs, each previously implicated in HU’s high‐affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUα(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure‐binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions.     

A Replication-Inhibited Unsegregated Nucleoid at Mid-Cell Blocks Z-Ring Formation and Cell Division Independently of SOS and the SlmA Nucleoid Occlusion Protein in Escherichia coli

Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. To investigate the nature of this coordination, the effects of inhibiting replication on Z-ring formation and cell division were tested in both synchronized and exponentially growing cells with only one replicating chromosome. When replication elongation was blocked by hydroxyurea or nalidixic acid, arrested cells contained one partially replicated, compact nucleoid located mid-cell. Cell division was strongly inhibited at or before the level of Z-ring formation. DNA cross-linking by mitomycin C delayed segregation, and the accumulation of about two chromosome equivalents at mid-cell also blocked Z-ring formation and cell division. Z-ring inhibition occurred independently of SOS, SlmA-mediated nucleoid occlusion, and MinCDE proteins and did not result from a decreased FtsZ protein concentration. We propose that the presence of a compact, incompletely replicated nucleoid or unsegregated chromosome masses at the normal mid-cell division site inhibits Z-ring formation and that the SOS system, SlmA, and MinC are not required for this inhibition.     

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.     

New subtilisin-trypsin inhibitors produced by Streptomyces: primary structures and their relationship to other proteinase inhibitors from Streptomyces

Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence analysis of the intact SIL proteins and peptides inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.     

The Bacterial Chromatin Protein HupA Can Remodel DNA and Associates with the Nucleoid in Clostridium difficile

The maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA and modulate its conformation, and by doing so affect a variety of cellular processes. No bacterial chromatin proteins of Clostridium difficile have been characterized to date.Here, we investigate aspects of the C. difficile HupA protein, a homologue of the histone-like HU proteins of Escherichia coli. HupA is a 10-kDa protein that is present as a homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. difficile. It binds to the DNA without a preference for the DNA G + C content. Upon DNA binding, HupA induces a conformational change in the substrate DNA in vitro and leads to compaction of the chromosome in vivo.The present study is the first to characterize a bacterial chromatin protein in C. difficile and opens the way to study the role of chromosomal organization in DNA metabolism and on other cellular processes in this organism.     

Properties of a membrane-attached form of the folded chromosome of Escherichia coli

Two types of DNA-containing particles are released from lysozyme-produced Escherichia coli spheroplasts after gentle lysis with non-ionic detergents in 1.-0 m-NaCl. Lysis at 25 °C releases the folded chromosomes (1300 S to 2200 S particles). Lysis at 10 °C results in faster sedimenting structures (3000 S to 4000 S). Both types of particles coexist in extracts of cells lysed at intermediate temperatures, i.e. 15 °C.The 3000 S to 4000 S particles are folded chromosomes attached to membrane fragments; they contain membrane proteins and phospholipids in addition to the folded DNA and nascent RNA chains. Incubation of the membrane-attached chromosomes with 1% Sarkosyl releases the folded chromosomes; this Sarkosyl treatment removes the membrane proteins and phospholipids, and halves the sedimentation velocity of the particles, but has no effect on the folded DNA and nascent RNA chains.Membrane-attached chromosomes cannot be isolated from amino acid-starved cells which have completed their rounds of DNA replication; all of the DNA then appears as released folded chromosomes. After resumption of protein synthesis, chromosome attachment to the membrane precedes the initiation of DNA replication. Controls strongly suggest that the changes observed, i.e. the attachment and release from the membrane of the folded chromosome, are related to the act of DNA replication itself.     

A simple autoradiographic method for investigating long range chromosome substructure: size and number of DNA molecules in isolated nucleoids of Escherichia coli.

A method is described for gently dissociating large DNA-protein complexes and for visualizing and quantitating the substructures by autoradiography. Using this technique, it is shown that nucleoids isolated from exponentially growing Escherichia coli (mean generation time = 35 min) contain on average 2.8 genome equivalents of DNA and that this nucleoid can be dissociated by deproteinization into two substructures having on average 1.4 genome equivalents. This result is correlated with previous sedimentation studies on the unfolded nucleoid DNA to explain prior inconsistencies. Scanning electron microscopy studies demonstrate that the shape and size of the isolated nucleoid is consistent with the proposed subunit structure of the in vivo nucleoid.