Changes in the fine structure of microbial cells induced by chaotropic salts

The electron microscopic examination of the thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37 degrees C for 3-5 h or at 100 degrees C for 5-6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

Fluorescence Microscopic Study of Microorganisms Treated with Chaotropic Agents

The yeasts Saccharomyces cerevisiae and Pichia pastoris and the bacteria Micrococcus luteus, Bacillus subtilis, and Anaerobacter polyendosporus have been treated with the chaotropic agents guanidine hydrochloride and guanidine thiocyanate and certain detergents and studied using fluorescence microscopy. Studies with the use of fluorochromes that can selectively stain nucleic acids (diamidino-2-phenylindole (DAPI), propidium iodide, and acridine orange) show that treatment of the bacterial and yeast cells at 37°C for 3–5 h induces a release of DNA from the cytoplasm and its accumulation in the cellular zone, known as ectoplasm, located between the cell wall and the remainder of the cytoplasm (called endoplasm) in the form of one or several large granules. After treating the cells with the chaotropic agents at 100°C for 5–6 min, the DNA is diffusively distributed over the ectoplasm. The fluorochromes used do not allow the detection of RNA. These findings are in agreement with previous data obtained from electron microscopic study of thin cell sections. After 33 PCR cycles, a considerable portion of DNA leaves the cells; as a result, they show a low level of diffusive fluorescence when stained with DAPI. When endospores of B. subtilis are treated with the chaotropic agents, they become highly permeable to the fluorochromes. Fluorescence microscopic study of such endospores shows that they contain DNA in the central part of their cores.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 505–510.Original Russian Text Copyright © 2005 by Duda, Danilevich, Akimov, Suzina, Dmitriev, Shorokhova.     

High Frequency Conjugative Mobilization Accomplished by a Natural Bacillus subtilisStrain Carrying a Large Plasmid

Conjugative properties of the strain Bacillus subtiliscarrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis168 strain with a frequency of approximately 10^–2. The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugative mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37°C, but conjugation also proceeded at lower temperatures, up to 21°C.     

Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon

Summary Seven mutations leading to riboflavin overproduction inBacillus subtilis were found to be linked to the markerdnaF133 (145° on theB. subtilis genetic map) by transformation. Cotransfer indexes (42.5%–61.7%) suggest that theribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26 –ts-6 –dnaF133 –ribC –recA1.     

Relaxation of supercoiled DNA associated with induction of heat shock proteins in Escherichia coli

Bacillus subtilis 168 is known to possess two thymidylate synthase (TSase; EC 2.1.1.45) genes: thyA and thyB. thyB encodes a thermosensitive TSase (inactivated at 46° C) which, in wild-type cells, accounts for only 5–8% of the total cellular TSase activity. In order to investigate the thermal lability of TSaseB we have analyzed the thyB genes of B. subtilis 168 and of an unrelated strain B. subtilis ATCC6633, which is shown here to have a temperature-resistant TSaseB. This conclusion is supported by the frequency of appearance of spontaneous Thy^– mutants at 37° C and 46° C, and by the analysis of clones containing the thyB genes from the two strains. The nucleotide sequences of these two thyB genes were compared.     

Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: Application in PCR

The procedure of obtaining DNA-containing cell envelopes (“micromummies”) of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.     

The effect of temperature on the survival of microorganisms in a deep space vacuum

A Space Molecular Sink Research Facility (Molsink) was used to evaluate the ability of microorganisms to survive the vacuum of outer space. this facility could be programmed to simulate flight spacecraft vacuum environments at pressures in the 10^–10 torr range and thermal gradients (30 to 60°C) closely associated to surface temperatures of inflight spacecraft.Initial populations ofStaphylococcus epidermidis and aMicrococcus sp. were reduced approximately 1 log while exposed to –105 and 34°C, and approximately 2 logs while exposed to 59°C for 14 days in the vacuum environment.Spores ofBacillus subtilis var.niger were less affected by the environment. Initial spore populations were reduced 0.2, 0.3, and 0.8 log during the 14-day vacuum exposure at –124, 34, and 59°C, respectively.This paper presents the results of one phase of research carried out at the Jet Propulsion Laboratory, California Institute of Technology, under Contract No. NAS 7-100, sponsored by the National Aeronautics and Space Administration.     

Isolation of a DNA-protein complex containing a single DNA fragment which is at or near the replication origin of the Bacillus subtilis chromosome

A chromosomal fragment containing purA, the nearest marker from the replication origin of the Bacillus subtilis chromosome, was highly purified as a complex containing at least proteins and being solubilized easily during cell lysis. The complex had a markedly higher sedimentation rate (70–120S) than the bulk of the solubilized DNA (40S). The electron microscopic observation showed the complex to be an aggregate of several DNA molecules with a local structure containing amorphous materials which stained black and bushes of RNA. This confirmed biochemical evidences suggesting that the complex is an intermolecular aggregate of the purA-DNA-protein-RNA complex.     

Nuclear and cell division in Bacillus subtilis Antibiotic-induced morphological changes

Incubation of Bacillus subtilis after outgrowth from spores in the presence of four different antibiotics in two different concentrations, showed that septation can occur without termination of nuclear division. Septation is then only partially uncoupled from the normal division cycle. Observations on location and development of mesosomes in the presence of the antibiotics, made in three-dimensional cell reconstructions, suggest that the mesosome plays a role in the normal coordination between nuclear and cell division, and may explain the partial independence between these two processes in B. subtilis.with technical assistance of Catherine J. SchaapThis work has been presented in part at the A.S.M. Conference on Bacilli: Biochemical Genetics, Physiology and Industrial Applications; 6–9 Aug, 1975, Ithaca, N.Y.     

Lactosucrose production by various microorganisms harboring levansucrase activity

Production of the artificial sweetener, lactosucrose, by various microorganisms containing levansucrase activity was investigated. Of the tested bacteria, Bacillus subtilis was the most effective producer using lactose as an acceptor and sucrose as a fructosyl donor. Lactosucrose production by this strain was optimal at pH 6.0 and 55 °C whereupon 181 g lactosucrose l^–1 was produced from 225 g lactose l^–1 and 225 g sucrose l^–1 in 10 h.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric C structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

Reproductive biology of Stictosiphonia hookeri (Rhodomelaceae,Rhodophyta) from Argentina,Chile, South Africa and Australia in laboratory culture

The red alga Stictosiphonia hookeri is epilithic in shaded habitats of the upper intertidal zone from 30 to 55° S. Thalli of this species from Argentina, Chile, South Africa and Australia, usually without reproductive structures when collected, all developed tetrasporangia in culture. Although good vegetative growth occurred in all nine isolates at 20–25 °C, 12:12 light: dark cycle, 10–30 µmol photons m^–2 s^–1, none reproduced in these conditions except one isolate from Australia. At 15 °C the four South African (34 °S) isolates developed tetrasporangial stichidia, and three completed a Polysiphonia-type life history. Gametophytes were unisexual or bisexual. At 15 °C one isolate from Chile (36 °S) formed tetrasporangia, but sporelings were not viable. At 10 °C isolates from Argentina and Chile (53 °S and 54 °S) formed tetrasporangia; however, only the Chile isolate completed a Polysiphonia-type life history with unisexual gametophytes. The temperature required to induce sporogenesis correlates with the range of water and air temperatures in the natural habitats of each isolate. In irradiances >50 µmol m^–2 s^–1 the thalli became yellow- brown within two weeks because of phycobiliprotein loss, but this did not impair growth or reproduction. The Argentina and Chile isolates were resistant to freezing in seawater for at least two days, showing no cell damage. The protein cuticle of the outer cell wall is repeatedly shed in culture. This may serve to minimize the attachment of epiphytes in the field.     

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment. Correspondence to: S. Shimada     

Effect of septum-initiation mutations on sporulation and competent cell formation in Bacillus subtilis

Summary Sporulation and competent cell formation have been studied in four Bacillus subtilis strains, carrying septum-initiation mutations of different loci, div-31, div-341, div-12 and div-355 which exhibit filamentous growth at 45° C. The div-31 mutant was found to be defective in competence development at 30°–40°C whereas the div-12 mutant was affected only slightly. The div-341 and div-355 mutants showed lower competence, particularly at the higher temperatures. The four div mutant strains all showed poor sporulation at higher temperatures compared to the wild-type strain. We propose that some of the initial steps of septation are involved both in sporulation (possibly in forespore septum formation) and in competent cell formation and that these two processes share certain common features distinct from those in vegetative cell division.     

Soil Strain of Bacillus subtilis Harboring a Large Plasmid That Mediates High-Frequency Conjugal Mobilization

The ability of a soil strain of Bacillus subtilis harboring a large plasmid, p19, to mobilize a small staphylococcal plasmid, pUB110, was studied. The latter plasmid was transferred to the recipient cells of Bacillus subtilis168 at a high frequency (about 10^–2 per recipient cell) both on the filter surface and in liquid medium. Mobilization was initiated 40 min after the beginning of the contact between donor and recipient cells.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Alkaline serine proteinase and lectin isolation from the culture fluid of Bacillus subtilis

Bacillus subtilis strain 316 M was found to produce extracellular alkaline serine proteinase and lectin. The characteristics of proteinase and lectin accumulation during the growth of the producer organism were found to be similar. The maxima of proteolytic and lectin activities were close and observed at 16 h and 14 h of B. subtilis 316 M batch cultivation, respectively. Alkaline serine proteinase was purified by ion exchange chromatography directly from the culture fluid. Proteinase (eluate) purified 40-fold possessed 60–90 units/ml of caseinolytic activity and 240–320 units/ml of elastolytic activity. Eluate obtained after enzyme sorption on the ion exchanger was used for lectin isolation followed by ammonium sulphate precipiration. Lectin purified 12.3-fold was shown to have a high carbohydrate specificity to N-glycolylneuraminic, N-acetylneuraminic, N-acetylmuramic and d-galacturonic acids with minimal inhibiting concentrations of 2.5–7.5 mm. *** DIRECT SUPPORT *** AG903053 00002     

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25°C. Replacement of K⁺ with Na⁺ effectively prevented the lysis by SCN⁻. K⁺ salts of NO₃⁻, Br⁻, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN⁻ than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN⁻ is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na⁺ observed here was considered to be manifested by specific interactions of Na⁺ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.     

Conjugal Plasmid Transfer in Bacillus subtilis under Conditions of Soil Microcosms

Conjugal transfer of the small plasmid pUB110 betweenBacillus subtilis strains was studied under conditions of microcosms with sterile and nonsterile soil. Plasmid transfer proved to be possible after soil inoculation with vegetative partner cells or with their spores. Plasmid transfer occurred at temperatures of 30 and 22–23°C.     

Hydroxylation of daidzein by CYP107H1 from Bacillus subtilis 168

CYP107H1, from Bacillus subtilis 168 known as fatty acid hydroxylase, showed the ortho-specific hydroxylation activity to daidzein, when coupled to the putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida as the redox partners. The electron transfer system of the three proteins was constructed in Escherichia coli BL21 (DE3) system using the two plasmids containing different selection markers. The daidzein hydroxylation was demonstrated with recombinant whole cell and in vitro system using the artificial redox partner for electron transfer. The identification of the hydroxylation reaction yielding 7,3′,4′-trihydroxyisoflavone was elucidated using gas chromatography mass spectrometry (GC–MS). This oxidizing activity of CYP107H1 towards daidzein represents the new hydroxylation of aromatic compound as substrate.