Salt-dependent and protein-concentration-dependent changes in the solution structure of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis.

The solution structure of the histone-like DNA-binding protein, HBsu, from Bacillus subtilis in 2 mM sodium cacodylate, pH 7.5, is sensitive to the ionic strength of the buffer. This was shown by circular dichroism measurements at different concentrations of sodium chloride and potassium fluoride. The stability of HBsu is also influenced; at HBsu concentrations of about 0.1 mg.ml-1, melting temperatures of 32 degrees C and 55 degrees C were found in the absence of potassium fluoride and in the presence of 0.5 M potassium fluoride, respectively, exhibiting drastic ionic-strength-dependent differences in the temperature-induced unfolding of HBsu. Furthermore, at low ionic strength, circular dichroism spectra vary markedly depending on the HBsu concentration in the approximate range 0.2-3 mg.ml-1. Such protein-concentration-dependent differences in the spectra were not observed in the presence of 0.5 M potassium fluoride. Very similar circular dichroism spectra of HBsu and the histone-like DNA-binding protein of Bacillus stearothermophilus (HBst) at high ionic strength, indicate comparable structures of both proteins under these conditions. Estimation of the secondary structure content from the circular dichroism spectra yields data which are in satisfactory agreement with the values obtained from the crystal structure of HBst. Transition temperatures of 45 degrees C and 61 degrees C were found in differential scanning calorimetric measurements performed with HBsu in potassium-fluoride-free buffer and in the presence of 0.5 M potassium fluoride, respectively. The thermodynamic data point to the melting of native HBsu dimers into two denatured monomers.     

Depletion of Bacillus subtilis histone-like protein, HBsu, causes defective protein translocation and induces upregulation of small cytoplasmic RNA

Small cytoplasmic RNA (scRNA) is a metabolically stable homologue of mammalian SRP RNA that contains an Alu-like domain. The Bacillus subtilis histone-like protein HBsu can bind this domain. We demonstrate here that repressing the level of HBsu results in slow growth and the accumulation of precursor of beta-lactamase fusion proteins having the signal sequence of alkaline protease, penicillin binding protein 5* (PBP5*) or CGTase. The degree of the translocation defect varied among the various signal sequences tested. A pulse-chase experiment showed that processing the alpha-amylase signal sequence is significantly inhibited in HBsu-depleted cells. Northern blot analysis indicated that repressing the HBsu gene induces scRNA upregulation, indicating that the defective translocation of presecretory proteins is not due to a reduced scRNA level. The data presented here suggest that HBsu plays a pivotal role in SRP function rather than simply stabilizing the other SRP components such as scRNA.     

Mutational analysis of the nucleoid-associated protein HBsu of Bacillus subtilis

The essential nucleoid-associated protein HBsu of Bacillus subtilis comprises 92 residues, 20% of which are basic amino acids. To investigate the role of the residues located within the DNA-binding arm, the arginine residues R58 and R61 were changed to leucine, while lysine residues K80 and K86 were replaced by alanine. All altered proteins exhibited a reduction in DNA binding capacity, ranging from 10% to 30% of HBsu wild type DNA-binding ability. To investigate the physiological effect of these mutations in B. subtilis, the indigenous hbs gene was replaced by the mutated genes. B. subtilis strain PK20, which carries the HBsu mutation R58L which exhibits the lowest DNA binding ability in vitro, showed the strongest retardation of growth compared to the wild type. Furthermore, PK20 cells displayed an increased rate of cell lysis, diminished sporulation efficiency and a reduced level of negatively supercoiled DNA. These observations suggest that the DNA binding ability of HBsu DNA is important for growth and differentiation and influences DNA topology.     

Determination of DNA-binding parameters for the Bacillus subtilis histone-like HBsu protein through introduction of fluorophores by site-directed mutagenesis of a synthetic gene.

A synthetic gene encoding the histone-like DNA-binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20 mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild-type protein. N-terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli. Site-directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA-binding parameters by fluorescence spectroscopy. According to gel-retardation experiments, the mutant protein [Phe47----Trp]HBsu shows identical DNA binding to wild-type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47----Trp]HBsu binds double-stranded DNA with a dissociation constant in the micromolar range. Computer-assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47----Trp]HBsu appears to contact three or four base pairs of DNA. These results are in partial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.     

Bacillus subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA

Small cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a ternary complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA.     

Completed Bacillus subtilis nucleoid as a doublet structure.

When outgrowing spores of the temperature-sensitive dna initiation mutants of Bacillus subtilis, TsB134 and dna-1, were allowed to undergo a single round of replication by shifting to the restrictive temperature soon after its initiation, both segregating daughter nucleoids appeared as clearly defined doublet structures. The components of each doublet remained together as a discrete pair, even under conditions which resulted in the formation of deoxyribonucleic acid (DNA)-less cells. A doublet nucleoid was also observed at a high frequency when TsB134 spores were allowed to germinate and grow out in the complete absence of DNA synthesis at the permissive temperature. TsB134 spores were foud to contain the usual "haploid" amount of DNA. It is suggested that the doublet nucleoid reflects a folding of a single chromosome into two large domains which resolve from one another under conditions of cell extension in the absence of DNA synthesis.     

Purification and properties of the DNA-binding protein HPB12 from Bacillus subtilis nucleoid

We report the purification to homogeneity of a 12 KDa protein (HPB12) present in the nucleoids of Bacillus subtilis. From the purification data the abundance of the protein was estimated to about 20,000 monomers per cell. The HPB12 protein is heat-stable and acid-soluble and binds preferentially to supercoiled and linearized double-stranded DNAs. The binding of the protein to the supercoiled DNA occurs very rapidly and appears to be cooperative. Moreover, the complexes are extremely stable and do not dissociate after 90 min. These properties are consistent with a role of the HPB12 protein in the structure of the B. subtilis chromosome.     

FtsZ and nucleoid segregation during outgrowth of Bacillus subtilis spores.

Spores of a strain of Bacillus subtilis in which ftsZ was under the control of the spac promoter were allowed to germinate and grow out in the presence of increasing concentrations of isopropyl-beta-D-thiogalactopyranoside (IPTG). Over the IPTG concentration range of 0 to 10(-3) M, the level of FtsZ from the time when the first nucleoid segregations were occurring, measured in Western blot (immunoblot) transfer experiments, varied between 15 and 100% of that in the wild type. Septation was completely blocked (for at least several hours) when the amount of FtsZ was < 30% of the wild-type level. At all levels of ftsZ induction, the timing and rate of segregation of nucleoids following the first round of replication were unaltered. It is concluded that FtsZ has no direct role in nucleoid segregation in this situation.     

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.     

Growth kinetics of individual Bacillus subtilis cells and correlation with nucleoid extension

The growth rate of individual cells of Bacillus subtilis (doubling time, 120 min) has been calculated by using a modification of the Collins-Richmond principle which allows the growth rate of mononucleate, binucleate, and septate cells to be calculated separately. The standard Collins-Richmond equation represents a weighted average of the growth rate calculated from these three major classes. Both approaches strongly suggest that the rate of length extension is exponential. By preparing critical-point-dried cells, in which major features of the cell such as nucleoids and cross-walls can be seen, it has also been possible to examine whether nucleoid extension is coupled to length extension. Growth rates for nucleoid movement are parallel to those of total length extension, except possibly in the case of septate cells. Furthermore, by calculating the growth rate of various portions of the cell surface, it appears likely that the limits of the site of cylindrical envelope assembly lie between the distal tips of the nucleoid; the old poles show zero growth rate. Coupling of nucleoid extension with increase of cell length is envisaged as occurring through an exponentially increasing number of DNA-surface attachment sites occupying most of the available surface.     

Energized membrane may regulate nucleoid conformation in Bacillus subtilis

Abstract It was shown in a previous study, using a temperature-sensitive initiation mutant of Bacillus subtilis that DNA synthesis at the origin and terminus of replication occurred at cell poles. In the current study, it is demonstrated that cells specifically labelled at the origin or near the terminus, show a re-distribution of radioactivity when treated with the uncoupling agent, sodium azide. Since it is most likely that the DNA-cell wall attachment is mediated through membrane interactions, we now propose that the maintenance of this attachment is dependent on the energized state of the membrane.     

Proteins from the prokaryotic nucleoid. Interaction of nucleic acids with the 15 kDa Escherichia coli histone-like protein H-NS

The interaction between nucleic acids and Escherichia coli H-NS, an abundant 15 kDa histone-like protein, has been studied by affinity chromatography, nitrocellulose filtration and fluorescence spectroscopy. Intrinsic fluorescence studies showed that the single Trp residue of H-NS (position 108) has a restricted mobility and is located within an hydrophobic region inaccessible to both anionic and cationic quenchers. Binding of H-NS to nucleic acids, however, results in a change of the microenvironment of the Trp residue and fluorescence quenching; from the titration curves obtained with addition of increasing amounts of poly(dA)-poly(dT) and poly(dC)-poly(dG) it can be estimated that an H-NS dimer in 1.5 x SSC binds DNA with an apparent Ka approximately equal to 1.1 x 10(4) M-1.bp-1. H-NS binds to double-stranded DNA with a higher affinity than the more abundant histone-like protein NS(HU) and, unlike NS, prefers double-stranded to single-stranded DNA and DNA to RNA; both monovalent and divalent cations are required for optimal binding.     

The Bacillus subtilis HBsu protein modifies the effects of alpha/beta-type, small acid-soluble spore proteins on DNA

HBsu, the Bacillus subtilis homolog of the Escherichia coli HU proteins and the major chromosomal protein in vegetative cells of B. subtilis, is present at similar levels in vegetative cells and spores ( approximately 5 x 10(4) monomers/genome). The level of HBsu in spores was unaffected by the presence or absence of the alpha/beta-type, small acid-soluble proteins (SASP), which are the major chromosomal proteins in spores. In developing forespores, HBsu colocalized with alpha/beta-type SASP on the nucleoid, suggesting that HBsu could modulate alpha/beta-type SASP-mediated properties of spore DNA. Indeed, in vitro studies showed that HBsu altered alpha/beta-type SASP protection of pUC19 from DNase digestion, induced negative DNA supercoiling opposing alpha/beta-type SASP-mediated positive supercoiling, and greatly ameliorated the alpha/beta-type SASP-mediated increase in DNA persistence length. However, HBsu did not significantly interfere with the alpha/beta-type SASP-mediated changes in the UV photochemistry of DNA that explain the heightened resistance of spores to UV radiation. These data strongly support a role for HBsu in modulating the effects of alpha/beta-type SASP on the properties of DNA in the developing and dormant spore.     

Coordination of cell division and chromosome segregation by a nucleoid occlusion protein in Bacillus subtilis

A range of genetical and physiological experiments have established that diverse bacterial cells possess a function called nucleoid occlusion, which acts to prevent cell division in the vicinity of the nucleoid. We have identified a specific effector of nucleoid occlusion in Bacillus subtilis, Noc (YyaA), as an inhibitor of division that is also a nonspecific DNA binding protein. Under various conditions in which the cell cycle is perturbed, Noc prevents the division machinery from assembling in the vicinity of the nucleoid. Unexpectedly, cells lacking both Noc and the Min system (which prevents division close to the cell poles) are blocked for division, apparently because they establish multiple nonproductive accumulations of division proteins. The results help to explain how B. subtilis specifies the division site under a range of conditions and how it avoids catastrophic breakage of the chromosome by division through the nucleoid.     

Structure-function relationship and regulation of two Bacillus subtilis DNA-binding proteins, HBsu and AbrB

Microorganisms use a number of small basic proteins for organization and compaction of their DNA. By their interaction with the genome, these proteins do have a profound effect on gene expression, growth behavior, and viability. It has to be distinguished between indirect effects as a consequence of the state of chromosome condensation and relaxation that influence the rate of RNA polymerase action as represented by the histone-like proteins, and direct effects by specific binding of proteins to defined DNA segments predominantly located around promoter sequences. This latter class is represented by the transition-state regulators that are involved in integrating various global stimuli and orchestrating expression of the genes under their regulation for a better adaptation to changes in growth rate. In this article we will focus on two different but abundant DNA binding proteins of the gram-positive model organism Bacillus subtilis, the histone-like HBsu as a member of the unspecific and the transition state regulator AbrB as a member of specific classes of DNA binding proteins.     

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.     

The essential histone-like protein HU plays a major role in Deinococcus radiodurans nucleoid compaction

The nucleoid of radioresistant bacteria, including D .  radiodurans , adopts a highly condensed structure that remains unaltered after exposure to high doses of irradiation. This structure may contribute to radioresistance by preventing the dispersion of DNA fragments generated by irradiation. In this report, we focused our study on the role of HU protein, a nucleoid-associated protein referred to as a histone-like protein, in the nucleoid compaction of D. radiodurans. We demonstrate, using a new system allowing conditional gene expression, that HU is essential for viability in D. radiodurans . Using a tagged HU protein and immunofluorescence microscopy, we show that HU protein localizes all over the nucleoid and that when HU is expressed from a thermosensitive plasmid, its progressive depletion at the non-permissive temperature generates decondensation of DNA before fractionation of the nucleoid into several entities and subsequent cell lysis. We also tested the effect of the absence of Dps, a protein also involved in nucleoid structure. In contrast to the drastic effect of HU depletion, no change in nucleoid morphology and cell viability was observed in dps mutants compared with the wild-type, reinforcing the major role of HU in nucleoid organization and DNA compaction in D. radiodurans .