An easy and rapid method for isolation of entire mycobacterial genome for application in pulsed-field gel electrophoresis

A DNA extraction suitable for mycobacterial lysis in gentle conditions compatible with genome analysis by pulsed-field gel electrophoresis is presented. Effects of preliminary treatments with SDS, Triton X-100, and hexane on mycobacterial outer layer were observed by electron microscopy. The most efficient procedure, performed on cells from liquid or solid medium, consisted of treatment by Triton X-100, agarose embedding of the cells, and further treatment with -amylase followed by lysozyme and SDS-proteinase K.     

Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. After cell harvest, the addition of detergent (0.1% Triton X-100) and a single 30 min freeze-thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high-throughput cultivation in microtiter plates, and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.     

Estimation of the growth rate of mixed ruminal bacteria from short-term DNA radiolabeling

A method based on 32P-labeling of DNA in short-term incubations was developed for estimating the growth rate of mixed rumen bacteria. A freeze/thaw procedure was optimized to quantitatively disrupt mixed rumen bacteria and extract bacterial DNA. The preliminary enzymatic lysis step, with lysozyme rather than proteinase K, sodium lauroyl sarcosine, and, to a lesser extent, sodium dodecyl sulfate (SDS) strongly improved cell disruption and DNA recovery rates. Sodium deoxycholate, CHAPS or Triton X-100 had no significant effect. Increasing the number of cycles or lowering the freezing temperature from -20 degrees C to -50 degrees C had no effect on DNA extraction efficiency while setting the thawing temperature at +60 degrees C rather than +37 degrees C slightly increased DNA yield but also increased its contamination with RNA. The method finally selected led to the lysis of at least 93% of cells and to the extraction of 85% of bacterial DNA. The kinetics of in vitro 32P incorporation into rumen bacteria DNA was then determined in batch incubations of strained rumen contents with no additional substrate. The curvilinear effects of the amount of 32P and the incubation time (5-15 min) on the DNA radioactivity were investigated by applying a Doehlert experimental design and fitting a second order polynomial model to data. The DNA radioactivity was linearly related to time (p<0.02) with other coefficients in the model being equal to zero (p>0.20). The incorporation of 32P into bacterial DNA was initiated approximately 70 s after the start of incubation. Taking into account the accuracy of scintillation counting, 10-15 min incubations, with 15 microCi 32P and 10 mL rumen contents per tube, appeared satisfactory for future studies.     

On-line cell lysis of bacteria and its spores using a microfluidic biochip

Optimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 10³ CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.     

The use of real-time PCR technology to assess the effectiveness of methods of DNA extraction from cultures of acidophilic chemolithotrophic microorganisms

Comparative evaluation of efficiency of several methods of DNA extraction from storage cultures of acidophilic chemolithotrophic microorganism communities isolated from sulfide ores of Shanuch ore deposit (Kamchatka peninsula) was conducted. DNA extraction methods in various combinations of physical (heating to 65–98°C, grinding with SiO₂ particles), enzymatic (treatment with lysozyme and proteinase K), and chemical (GuSCN, CTAB and KOH) treatments were tested. The evaluation of efficiency was performed using Real-time PCR. The best result was obtained for the combined method based on GuSCN lysis activity (lysis at 65°C) followed by purification with phenol and chloroform.     

Autoclave method for rapid preparation of bacterial PCR-template DNA

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.     

Halococcus sediminicola sp. nov., an extremely halophilic archaeon isolated from a marine sediment

A novel, red-pigmented and coccoid haloarchaeon, designated strain CBA1101^T, was isolated from a marine sediment. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CBA1101^T is most closely related to the genus Halococcus in the family Halobacteriaceae. Strain CBA1101^T had a highest 16S rRNA gene sequence similarity of 98.4 % with Halococcus dombrowskii DSM 14522^T, followed by 93.7–98.3 % with sequences of other type strains in the genus Halococcus. The RNA polymerase subunit B′ gene sequence similarity of strain CBA1101^T with that of Halococcus qingdaonensis JCM 13587^T is 89.5 % and lower with those of other members of the genus Halococcus. Strain CBA1101^T was observed to grow at 25–40 °C, pH 6.0–9.0 and in the presence of 15–30 % (w/v) NaCl, with optimal growth at 35–40 °C, pH 7.0 and with 20 % NaCl. The cells of strain CBA1101^T are Gram-negative and did not lyse in distilled water. The major polar lipids were identified as phosphatidylglyerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl diether, unidentified phospholipids and unidentified glycolipids. The genomic DNA G+C content was determined 66.0 mol%. The DNA–DNA hybridization experiment showed that there was less than 40 % relatedness between strain CBA1101^T and the reference species in the genus Halococcus. Based on this polyphasic taxonomic analysis, strain CBA1101^T is considered to represent a new species in the genus Halococcus, for which the name Halococcus sediminicola sp. nov. is proposed. The type strain is CBA1101^T (=JCM 18965^T = CECT 8275^T).     

Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by Triton X-100 for efficient production of d-malate

Pseudomonas pseudoalcaligenes can only form d-malate from maleate after incubation of the cells with a solvent or a detergent. The effect of the detergent Triton X-100 on d-malate production was studied in more detail. The longer the cells were incubated with Triton X-100, the higher was the d-malate production activity, until the maximal malease activity was reached. Incubation of P. pseudoalcaligenes cells with Triton X-100 also resulted in an increase in the protein concentration of the supernatant, indicating that cell lysis had occurred. The rate at which the d-malate production activity increased was dependent on the Triton X-100 concentration and on the cell density. Also the rate at which lysis occurred depended on the Triton X-100 concentration.     

Identification of polyhydroxyalkanoates in Halococcus and other haloarchaeal species

Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and ¹H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307^T, Halococcus saccharolyticus DSM 5350^T, Halococcus salifodinae DSM 8989^T, Halococcus dombrowskii DSM 14522^T, Halococcus hamelinensis JCM 12892^T, Halococcus qingdaonensis JCM 13587^T), Halorubrum sp. (Hrr. coriense DSM 10284^T, Halorubrum chaoviator DSM 19316^T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189^T, Natronococcus occultus DSM 3396^T) and Halobacterium noricense DSM 9758^T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757^T. Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ß-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times.     

Adsorption of surfactants on a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain and the effect on cell surface lypohydrophilic property

The adsorption behavior of five surfactants, cetyltrimethylammonium bromide (CTAB), Triton X-100, Tween 80, sodium dodecyl sulfate (SDS), and rhamnolipid, on a Pseudomonas aeruginosa strain and the effect of temperature and ionic strength (IS) on the adsorption were studied. The change of cell surface lypohydrophilic property caused by surfactant adsorption was also investigated. The results showed that the adsorption kinetics of the surfactants on the cell followed the second-order law. CTAB adsorption was the fastest one under the experimental conditions, and it took longest for SDS adsorption to equilibrate because of electric repulsion. The adsorption of Triton X-100 and Tween 80 was characterized by short equilibration time, and rhamnolipid adsorption reached equilibrium in about 90 min. The adsorption isotherms of all the surfactants on the bacterium fitted Freundlich equation well, but the adsorption capacity and mode were variations for the surfactants as indicated by k and n parameters in the equations. The adsorption mode for all the surfactants except SDS is probably hydrophilic interaction because the adsorption totally turned the cell surface to be more hydrophobic. Neither the temperature nor the IS had significant effect on CTAB adsorption, but higher IS significantly enhanced SDS adsorption and modestly strengthened adsorption of Triton X-100, Tween 80, and rhamnolipid. Higher temperature strengthened adsorption of SDS but weakened the adsorption of Triton X-100, Tween 80, and rhamnolipid.     

Trypanosoma brucei: activities and subcellular distribution of glycolytic enzymes from differently disrupted cells

The effect of disruption procedure on the subcellular distribution and the activities of 11 enzymes catalyzing the glycolytic pathway in Trypanosoma brucei has been studied. The activities of the enzymes varied with the lytic procedure used. Maximum specific enzyme activity values were obtained after treatment with saponin whereas digitonin treatment gave the lowest results. The intracellular location of the enzymes was examined by means of differential centrifugation following cell lysis with saponin, Triton X-100, digitonin, or by freezing and thawing. Irrespective of the method of cell lysis employed, the six enzymes, hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, glycerol phosphate dehydrogenase, and glycerokinase, were particulate. Of the remaining 5 enzymes, digitonin liberates only phosphoglycerate mutase (partially); saponin or Triton X-100 liberates phosphoglucose isomerase, phosphoglycerate mutase, enolase, and pyruvate kinase but not glyceraldehyde 3-phosphate dehydrogenase; freezing and thawing acts like saponin or Triton X-100 except that it fails to liberate phosphoglucose isomerase, while cell grinding with silicon carbide liberates only glyceraldehyde phosphate dehydrogenase (partially), phosphoglycerate mutase, enolase, and pyruvate kinase. The relative maximal activities of the enzymes suggest that the rate-limiting steps in glycolysis in T. brucei are the reactions catalyzed by aldolase and phosphoglycerate mutase.     

Isolating conifer DNA: A superior polysaccharide elimination method

Genomic DNA was isolated from frozen needles of maturePinus radiata clones using a modified extraction technique incorporating cetyltrimethylammonium bromide (CTAB) for cell lysis. A high sodium chloride concentration (2 M) was used at 2 stages of the extraction procedure to eradicate polysaccharides, yielding pure genomic DNA suitable for restriction enzyme digestion and PCR amplification. Extractions were scaled down to suit 1.5-mL Eppendorf tubes, allowing easier handling and enhanced sterility.     

Comparative quantitative proteomics of prochlorococcus ecotypes to a decrease in environmental phosphate concentrations

Background

Goa is a coastal state in India and salt making is being practiced for many years. This investigation aimed in determining the culturable haloarchaeal diversity during two different phases of salt production in a natural solar saltern of Ribandar, Goa. Water and sediment samples were collected from the saltern during pre-salt harvesting phase and salt harvesting phase. Salinity and pH of the sampling site was determined. Isolates were obtained by plating of the samples on complex and synthetic haloarchaeal media. Morphology of the isolates was determined using Gram staining and electron microscopy. Response of cells to distilled water was studied spectrophotometrically at 600nm. Molecular identification of the isolates was performed by sequencing the 16S rRNA.

Results

Salinity of salt pans varied from 3-4% (non-salt production phase) to 30% (salt production phase) and pH varied from 7.0-8.0. Seven haloarchaeal strains were isolated from water and sediment samples during non-salt production phase and seventeen haloarchaeal strains were isolated during the salt production phase. All the strains stained uniformly Gram negative. The orange-red acetone extract of the pigments showed similar spectrophotometric profile with absorption maxima at 393, 474, 501 and 535 nm. All isolates obtained from the salt dilute phase were grouped within the genus Halococcus. This was validated using both total lipid profiling and 16S rRNA data sequencing. The isolates obtained from pre-salt harvesting phase were resistant to lysis. 16S rRNA data showed that organisms belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained during the salt concentrated phase. The isolates obtained from salt harvesting phase showed varied lysis on suspension in distilled water and /or 3.5% NaCl.

Conclusion

Salterns in Goa are transiently operated during post monsoon season from January to May. During the pre-salt harvesting phase, all the isolates obtained belonged to Halococcus sp. During the salt harvesting phase, isolates belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained. This study clearly indicates that Halococcus sp. dominates during the low salinity conditions.     

Effects of Detergents on Lysis of Micrococcus lysodeikticus by Lysozyme

The effects of detergents on the lysozyme-catalyzed hydrolysis of Micrococcus lysodeikticus cells were investigated by changing the concentration of Na-phosphate buffer and pH in the presence or absence of sucrose. Also, a parallel study of the hydrolysis of glycolchitin by lysozyme was conducted and compared to the lytic reaction. Electron microscopy was utilized to follow the changes in cell morphology during the various treatments.

None of the detergents changed turbidity of the cell suspension. However, they did affect the change in turbidity during lysis in unique ways. SDS, which is an anionic detergent, inhibited lysozyme activity and its addition to the reaction mixture caused a rapid and large decrease in the turbidity. Brij 35 and Triton X-100, which are non-ionic detergents, did not inhibit lysozyme activity, but their presence in the reaction mixture changed the rate of turbidity change. Apparently non-ionic detergents disrupt only the protoplast, while anionic detergents disrupt both the protoplast and the damaged cell. The lytic mechanism of M. lysodeikticus by lysozyme was discussed in detail.     

Occurrence of Halococcus spp. in the nostrils salt glands of the seabird Calonectris diomedea

The nostrils of the seabird Calonectris diomedea are endowed with a salt-excreting gland that could produce a suitable environment for the colonization of extreme halophilic prokaryotes. We have studied in this organ the presence of extreme halophiles by means of culturing techniques. We could easily cultivate members of haloarchaea, and all cultures studied were identified as members of one of the two species Halococcus morrhuae and Hcc. dombrowskii. In order to reveal the diversity of these colonizers, we undertook a taxonomic study. Altogether, the results indicated that members of the genus Halococcus may constitute a part of the natural epizootic microbiota of C. diomedea, and that they exhibit such an important degree of taxonomic variability that appeals for a pragmatic species definition. This seabird nests in the west Mediterranean coasts, but its migratory habits, reaching locations as distant from the Mediterranean as the South Atlantic, may help in the dispersal mechanisms of haloarchaea through the Earth’s surface.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

Transforming activity of plasmid and chromosomal DNA inEscherichia coli

An auxotrophic strain ofEscherichia coli with therecB recC sbcB genotype was transformed by chromosomal DNA of the prototrophic strain and by plasmid DNA carrying genes for antibiotic resistance (R1drd 19). The donor plasmid DNA obtained by cell lysis in the presence of Triton X-100 and subsequent centrifugation in a caesium chloride-ethidium bromide gradient was shown to have a circulaf molecule and to retain its completeness after penetration into the recipient. Experiments with mixtures or plasmid and chromosomal DNA indicate a competition between these two DNA types during the transformation reaction in the given system.     

A simple procedure for the solubilization of NADH-cytochrome b5 reductase

NADH-cytochrome b₅ reductase has been solubilized by extraction of rabbit liver microsomes with 1 potassium phosphate buffer (pH 7.4), and has been purified to comparable purity with the Triton X-100-solubilized enzyme. Gel electrophoresis indicated an apparent molecular weight of 33,000 for both phosphate buffer-extracted and Triton X-100-solubilized enzymes. Phosphate buffer extraction provides a simple mild procedure for the extraction of NADH-cytochrome b₅ reductase that avoids detergents or proteolytic agents.     

Characterization of essential enolase in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we characterized the essentiality of enolase for growth of Staphylococcus aureus in vitro by using a TetR-regulated antisense RNA expression technology. The induced enolase antisense RNA dramatically decreased the production of enolase, which in turn inhibited the growth of S. aureus. In addition, we found that the down-regulation of eno expression can effectively inhibit Triton X-100-induced lysis and alleviate penicillin-caused cell lysis. To further confirm the specific effect of enolase on autolysis, we constructed an enolase over-expression system and demonstrated that the over-expression of enolase enhances both Triton X-100 and penicillin-induced cell lysis without increasing cell growth rate. We also performed hydrolase induced autolysis and zymographic assays and found that enolase had no impact on either bacterial sensitivity to hydrolase or hydrolase activity. Moreover, we found that the down-regulating expression of enolase selectively increased bacterial sensitivity to phosphomycin. Taken together, the above results suggest that the enolase is essential for S. aureus and involved in the process of bacterial autolysis.     

A simplified method for assay of hydrogenase activities of H<Subscript>2</Subscript> evolution and uptake in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis>

Assay of hydrogenase activity pertaining to H₂ production needs anaerobic conditions. To establish a simplified method for assay of hydrogenase activities by using intact cells of Enterobater aerogenes, different chemicals capable of enhancing the cell-wall permeability to electron mediators were examined. As a result, Triton X-100 and CTAB were found to be appropriate for H₂ uptake and evolution activities of the intact cells, respectively. This method enabled H₂ uptake and evolution activities of the intact cells to be easily detected. This is also the first report of the presence of H₂ uptake hydrogenase activity in E. aerogenes.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 12 April 2005 and 17 May 2005