Growth phase dependent changes in the structure and protein composition of nucleoid in Escherichia coli

The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, structures as well as protein/DNA composition of isolated nucleoids were then compared under various growth phases. The nucleoid structures were found to undergo changes during the cell growth; i. e., the nucleoid structure in the stationary phase was more tightly compacted than that in the exponential phase. In addition to factor for inversion stimulation(Fis), histone-like nucleoid structuring protein(H-NS), heat-unstable nucleoid protein(HU) and integration host factor(IHF) here we have identified, three new candidates of E. coli nucleoid, namely DNA-binding protein from starved cells(Dps), host factor for phage Qβ(Hfq) and suppressor of td- phenotype A(Stp A). Our results reveal that the major components of exponential phase nucleoid are Fis, HU, H-NS, Stp A and Hfq, while Dps occupies more than half of the stationary phase nucleoid. It has been known for a while that Dps is the main nucleoid-associated protein at stationary phase. From these results and the prevailing information, we propose a model for growth phase dependent changes in the structure and protein composition of nucleoid in E. coli.     

DNA-synthesizing activity of the nucleoid protein core in Escherichia coli

Nucleoids obtained from E. coli cells by extraction with 1 M NaCl and detergents containing solution were further extracted with 2 M NaCl. From these samples, that contain only tightly bound proteins, fractions of protein core and peripheral nucleoprotein were obtained. It is shown that DNA synthesis proceeds mainly in the core structures. We have found that DNA polymerase I, which is bound with DNA nucleoid loops and with the above mentioned core structures, is not dissociating in 2M NaCl solution.     

Dimerization of the RelA protein of Escherichia coli.

The RelA protein of Escherichia coli is a ribosome-associated (p)ppGpp synthetase that is activated by amino acid deprivation. It was recently reported that the activity of RelA is regulated by oligomerization mediated by the C-terminal domain of RelA. The oligomerization of RelA is further characterized in this study. The C-terminal domain consisting of amino acids 455-744, designated 'RelA, formed homooligomers as well as heterooligomers with RelA as demonstrated by copurification of RelA and 'RelA and by an affinity blotting assay. Glutaraldehyde-induced cross-linking indicated that the oligomer was a dimer. The functional analysis of 'RelA was based on a combination of yeast two-hybrid analysis, the determination of the effects of overexpression of 'RelA derivatives on the stringent response, and the cellular localization of the overexpressed 'RelA derivatives. These studies indicated that two regions, designated 'RelA-1 (amino acids 455-538) and 'RelA-2 (amino acids 550-682), were involved in dimerization. The involvement of one of these two regions, RelA-2, is consistent with a previous site-directed mutagenesis study. In addition to dimerization, 'RelA-2 apparently contained the main ribosome-binding domain of RelA. The third region, 'RelA-3 (amino acids 682-744), was not involved in either dimerization or ribosome binding. The overexpression of 'RelA-1 and 'RelA-2, but not 'RelA-3, inhibited the stringent response. These results support the previously proposed model which suggests a role for oligomerization in the regulation of (p)ppGpp synthetase.     

A DNA‐promoted amyloid proteinopathy in Escherichia coli

Protein amyloids arise from the conformational conversion and assembly of a soluble protein into fibrilar aggregates with a crossed β‐sheet backbone. Amyloid aggregates are able to replicate by acting as a template for the structural transformation and accretion of further protein molecules. In physicochemical terms, amyloids arguably constitute the simplest self‐replicative macromolecular assemblies. Similarly to the mammalian proteins PrP and α‐synuclein, the winged‐helix dimerization (WH1) domain of the bacterial, plasmid‐encoded protein RepA can assemble into amyloid fibres upon binding to DNA in vitro. Here we report that a hyper‐amyloidogenic functional variant (A31V) of RepA, fused to a red fluorescent protein, causes an amyloid proteinopathy in Escherichia coli with the following features: (i) in the presence of multiple copies of the specific DNA sequence opsp, WH1(A31V) accumulates as cytoplasmatic inclusions segregated from the nucleoid; (ii) such aggregates are amyloid in nature; (iii) bacteria carrying the amyloid inclusions age, exhibiting a fivefold expanded generation time; (iv) before cytokinesis, small inclusions are assembled de novo and transferred to the daughter cells, in which transmission failures cure amyloidosis; and (v) in the absence of inducer DNA, purified cellular WH1(A31V) inclusions seed amyloid fibre growth in vitro from the soluble protein. RepA‐WH1 is a suitable bacterial model system for amyloid proteinopathies.     

Isolation of the Escherichia coli nucleoid

Numerous protocols for the isolation of bacterial nucleoids have been described based on treatment of cells with sucrose-lysozyme-EDTA and subsequent lysis with detergents in the presence of counterions (e.g., NaCl, spermidine). Depending on the lysis conditions both envelope-free and envelope-bound nucleoids could be obtained, often in the same lysate. To investigate the mechanism(s) involved in compacting bacterial DNA in the living cell, we wished to isolate intact nucleoids in the absence of detergents and high concentrations of counterions. Here, we compare the general lysis method using detergents with a procedure involving osmotic shock of Escherichia coli spheroplasts that resulted in nucleoids free of envelope fragments. After staining the DNA with DAPI (4',6-diamidino-2-phenylindole) and cell lysis by either isolation procedure, free-floating nucleoids could be readily visualized in fluorescence microscope preparations. The detergent-salt and the osmotic-shock nucleoids appeared as relatively compact structures under the applied ionic conditions of 1 M and 10 mM, respectively. RNase treatment caused no dramatic changes in the size of either nucleoid.     

Mechanism of chromosome compaction and looping by the Escherichia coli nucleoid protein Fis

Fis, the most abundant DNA-binding protein in Escherichia coli during rapid growth, has been suspected to play an important role in defining nucleoid structure. Using bulk-phase and single-DNA molecule experiments, we analyze the structural consequences of non-specific binding by Fis to DNA. Fis binds DNA in a largely sequence-neutral fashion at nanomolar concentrations, resulting in mild compaction under applied force due to DNA bending. With increasing concentration, Fis first coats DNA to form an ordered array with one Fis dimer bound per 21 bp and then abruptly shifts to forming a higher-order Fis-DNA filament, referred to as a low-mobility complex (LMC). The LMC initially contains two Fis dimers per 21 bp of DNA, but additional Fis dimers assemble into the LMC as the concentration is increased further. These complexes, formed at or above 1 microM Fis, are able to collapse large DNA molecules via stabilization of DNA loops. The opening and closing of loops on single DNA molecules can be followed in real time as abrupt jumps in DNA extension. Formation of loop-stabilizing complexes is sensitive to high ionic strength, even under conditions where DNA bending-compaction is unaltered. Analyses of mutants indicate that Fis-mediated DNA looping does not involve tertiary or quaternary changes in the Fis dimer structure but that a number of surface-exposed residues located both within and outside the helix-turn-helix DNA-binding region are critical. These results suggest that Fis may play a role in vivo as a domain barrier element by organizing DNA loops within the E. coli 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.     

Characterization of the Escherichia coli YedU protein as a molecular chaperone

We have cloned, purified to homogeneity, and characterized as a molecular chaperone the Escherichia coli YedU protein. The purified protein shows a single band at 31 kDa on SDS-polyacrylamide gels and forms dimers in solution. Like other chaperones, YedU interacts with unfolded and denatured proteins. It promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation and prevents the aggregation of citrate synthase under heat shock conditions. YedU forms complexes with the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin. In contrast to DnaK/Hsp70, ATP does not stimulate YedU-dependent citrate synthase renaturation and does not affect the interaction between YedU and unfolded proteins, and YedU does not display any peptide-stimulated ATPase activity. We conclude that YedU is a novel chaperone which functions independently of an ATP/ADP cycle.     

HU-GFP and DAPI co-localize on the Escherichia coli nucleoid

The heterodimeric HU protein, one of the most abundant DNA binding proteins, plays a pleiotropic role in bacteria. Among others, HU was shown to contribute to the maintenance of DNA superhelical density in Escherichia coli. By its properties HU shares some traits with histones and HMG proteins. More recently, its specific binding to DNA recombination and repair intermediates suggests that HU should be considered as a DNA damage sensor. For all these reasons, it will be of interest to follow the localization of HU within the living bacterial cells. To this end, we constructed HU-GFP fusion proteins and compared by microscopy the GFP green fluorescence with images of the nucleoid after DAPI staining. We show that DAPI and HU-GFP colocalize on the E. coli nucleoid. HU, therefore, can be considered as a natural tracer of DNA in the living bacterial cell.     

Impairment of nucleoid segregation and cell division at high osmolarity in a strain of Escherichia coli overproducing the chaperone DnaK

Abstract Fourteen species of ciliates, seven of which are new, were found living in a sample of anoxic water collected from a small lake in Spain. The species belong to all six orders in which anaerobic ciliates have been described and they include the first anaerobic representatives of the order Prostomatida. This surprising diversity is probably sustained because it embraces all ciliate feeding types, and because protozoa are the only important consumers of the diversity of microbes in anoxic habitats. Six of the anaerobic ciliate species have aerobic congeners; this strengthens the contention that anaerobic ciliates evolved independently from aerobes belonging to several taxonomic groups.     

The Escherichia coli DjlA and CbpA proteins can substitute for DnaJ in DnaK-mediated protein disaggregation

The DnaJ (Hsp40) protein of Escherichia coli serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other E. coli proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in dnaJ mutants both CbpA and DjlA are essential for growth at temperatures above 37 degrees C, their in vivo role is poorly understood. Here we show that in a dnaJ mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42 degrees C.     

Age‐dependent racemization of serine residues in a human chaperone protein

Racemization is one of the most abundant modifications in long‐lived proteins. It has been proposed that the accumulation of such modifications over time could lead to changes in tissues and ultimately human age‐related diseases. Serine is one of the main amino acids involved in racemization; however, the site of D‐Ser in any aged protein has yet to be reported. In this study, racemization of two residues, Ser 59 and Ser 62, has been demonstrated in an unstructured region of the small heat shock protein, αA‐crystallin. αA‐crystallin is also the most abundant structural protein in the human lens. D‐Ser increased linearly with age in normal lenses, until it accounted for approximately 35% of the Ser at both sites by the age of 75 years. In agreement with a possible role in human age‐related disease, levels were significantly higher in cataract lenses. It is likely that such prevalent age‐related changes contribute to the denaturation of α‐crystallin, and therefore its ability to act as a chaperone. Racemization of amino acids, such as serine, in flexible regions of long‐lived proteins, could be associated with the development of human age‐related conditions such as cataract.     

Isolation and characterization of nucleoid proteins from Escherichia coli

Summary Of the molecular species of proteins associated with the nucleoids of Escherichia coli cells, those with relatively high affinity to bind to DNA were isolated and characterized. Seven classes of nucleoid proteins with molecular weights of 9,000, 17,000 (two molecular species), 22,000, 24,000, 27,000 and 28,000 were isolated at more than 90% purity or were partially purified. On the basis of its amino acid composition and other chemical properties, the 9,000 dalton protein was identified as HLP II (or HU protein or BH2) (Pettijohn 1982: Rouvière-Yaniv and Gros 1975; Varshavsky et al. 1978). The 17 K protein consisted of two molecular species and one of these, 17 K (a) protein, seemed to be identical with HLPI (or protein 1 or BH1) reported previously (Pettijohn 1982; Varshavsky et al. 1977; Varshavsky et al. 1978). The 26 K protein was identical to the 22 K protein (Kishi et al. 1982). The 27 K protein showed immunological cross-reactivity with the antibody for histone H2A and was thus identified as the H protein reported previously (Hübscher et al. 1980). Two basic proteins, 9 K and 17 K(a), showed relatively high binding affinities to DNA, while the 28 K protein showed moderate binding affinity. The biological significance of these nucleoid proteins, which constitute a family of proteins participating in formation of the nucleoid structure, is discussed.     

Solution structure of the cysteine-rich domain of the Escherichia coli chaperone protein DnaJ

The solution structure of the cysteine-rich (CR) domain of Escherichia coli DnaJ has been solved by NMR methods. The structure of a 79 residue CR domain construct shows a novel fold with an overall V-shaped extended beta-hairpin topology. The CR domain is characterized by four C-X-X-C-X-G-X-G sequence motifs that bind two zinc ions. Residues in these two zinc modules show strong similarities in the grouping of resonances in the (15)N-(1)H HSQC spectrum and display pseudo-symmetry of the motifs in the calculated structures. The conformation of the cysteine residues coordinated to the zinc ion resembles that of the rubredoxin-knuckle, but there are significant differences in hydrogen bonding patterns in the two motifs. Zinc (15)N-(1)H HSQC titrations indicate that the fold of the isolated DnaJ CR domain is zinc-dependent and that one zinc module folds before the other. The C-X-X-C-X-G-X-G sequence motif is highly conserved in CR domains from a wide variety of species. The three-dimensional structure of the E. coli CR domain indicates that this sequence conservation is likely to result in a conserved structural motif.     

Population length variability and nucleoid numbers in Escherichia coli

MOTIVATION: Cell sizes and shapes are a fundamental defining characteristic of all cellular life. In bacteria like Escherichia coli, the machinery that determines cell length is complex and interconnected, spanning extracellular cues, biosynthesis and cell division. Few tools exist to study cell lengths in a population. We have developed and tested three automated image analysis routines on growing E.coli cultures to simultaneously measure cell lengths and nucleoid numbers in populations of bacteria. We find population profiles changing with culture density-higher density of culture leads to fewer long cells. Additionally, lab strains mutant for recA show a correlation between the number of nucleoids and cell length. CONTACT: cathale@iiserpune.ac.in; chaitanya.athale@gmail.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

The looped domain organization of the nucleoid in histone-like protein defective Escherichia coli strains.

We have investigated the major Escherichia coli histone-like proteins (H-NS, HU, FIS, and IHF) as putative factors involved in the maintenance of the overall DNA looped arrangement of the bacterial nucleoid. The long-range architecture of the chromosome has been studied by means of an assay based on in vivo genomic fragmentation mediated by endogenous DNA gyrase in the presence of oxolinic acid. The fragmentation products were analysed by CHEF electrophoresis. The results indicate that in vivo a large fraction of the bacterial chromatin constitutes an adequate substrate for the enzyme. DNA fragments released upon oxo-treatment span a size range from about 1000 kb to a limit-size of about 50 kb. The latter value is in excellent agreement with the average size reported for bacterial chromosomal domains. The DNA gyrase-mediated fragmentation does not appear to be significantly altered in strains depleted in histone-like proteins as compared to an E. coli wild type strain. This suggests that these proteins may not represent critical determinants for the maintenance of the supercoiled loop organisation of the E. coli chromosome.     

Mitomycin-C-induced changes in the nucleoid of Escherichia coli K12

The influence of low concentrations of mitomycin-C on the structure of the envelope-free nucleoid was studied in several strains of Escherichia coli K12. The wild-type strain AB1157 uvr+ rec+ and 3 mitomycin-C-sensitive derivatives carrying mutations in the uvrA, uvrB and recA genes, were used. Treatment of the control strain with mitomycin-C, 0.5 microgram/ml, followed by incubation in drug-free medium resulted in the formation of a transient fast-sedimenting nucleoid with a sedimentation coefficient of 2200 S. A fraction of 25% of the nucleoids had attained the normal sedimentation coefficient of 1570 S 3 h after removal of mitomycin-C. With the uvr- strains, mitomycin-C induced a slow, almost linear increase in the S value of the envelope-free nucleoid. In these cases the S value continued to increase during post-incubation and was 2050 S 3 h after removal of the drug. Post-incubation of recA- cells resulted in loss of supercoiling, decrease in S value of the nucleoid and degradation of DNA. Results obtained with phase-contrast and electron microscopy were in good agreement with the hydrodynamic data.