Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.     

The cellular location and effect on nisin immunity of the NisI protein from Lactococcus lactis N8 expressed in Escherichia coli and L. lactis

Abstract Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cm^t-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 × 10³ transformants per μg DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 × 10² transformants per μg pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens.     

Transformation of Aspergillus alliaceus using the Aspergillus niger niaD gene encoding nitrate reductase

Abstract A heterologous transformation system for Aspergillus alliaceus based on the Aspergillus niger nitrate reductase structural gene ( niaD ) has been developed. Two mutants of A. alliaceus (M3 and M17), each carrying an niaD mutation were isolated by screening UV-irradiated cells for the inability to grow on nitrate as sole nitrogen source. Using plasmid pSTA 10, transformation frequencies of 4 and 200 per μg DNA respectively were obtained for these two strains. All the niaD ⁺ transformants tested were mitotically stable. Southern hybridisation analyses showed that the vector DNA sequences were present.     

Biological activity of monomeric Sendai virus HN glycoprotein

Abstract We have developed a transformation system for Streptomyces wadayamensis , a cephamycin C producer. 1−5 × 10⁹ protoplasts can be obtained when late logarithmic phase cultures of this microorganisms are incubated with 10 mg/ml of lysozyme. Polyethylene glycol-Ca²⁺-mediated transformation of these protoplasts yielded 10⁶ transformants per μg of pIJ702 or pIJ365 DNA.     

Transformation of group A streptococci by electroporation

Abstract The introduction, via electroporation, of free plasmid DNA into three strains of Streptococcus pyogenes is described. The method is very simple and rapid and efficiencies vary from 1 × 10³ to 4 × 10⁴ per μg of DNA. The method was also used to introduce an integrative plasmid and transformants were obtained, albeit at a somewhat lower frequency (2 × 10²). Some of the plasmids used in this study are derivatives of the Lactococcus lactis subsp. cremoris Wg2 plasmid pWV01. These broad host range vectors replicate in Gram-positives as well as Gram-negatives (viz. Escherichia coli ). Here we show that they also replicate in S. pyogenes and S. sanguis .     

Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation

Aims:  The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10²–10⁵ transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results:  MgCl₂ and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg²⁺ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg²⁺ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg²⁺. To maintain the stability of the cell membranes, Mg²⁺ was replaced with high concentrations of sucrose, from 272 to 408 mmol l⁻¹. With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 10⁶ transformants per microgram DNA.
Conclusions:  Mg²⁺ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg²⁺ is absent.
Significance and Impact of the Study:  A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg²⁺ can be used for other nuclease-producing organisms.     

Transformation of Streptococcus bovis protoplasts by plasmid DNA

M. MAREKOVÁ, V. KMET' AND P. JAVORSKÝ. 1996. The transformation and subsequent regeneration of ruminal strain Streptococcus bovis AO24/85 protoplasts by plasmid DNA was studied. The best stabilizer for regeneration of protoplasted cells was 5% sucrose in the regeneration medium and in the agar plates. Optimal concentration of polyethylene glycol 6000 in the transformation medium was 25% for both plasmids tested. Addition of Ca²⁺ and Mg²⁺ ions (2.5 mmol l^-1) to the transformation medium increased the proportion of regenerated cells. Transformation frequencies were 3 times 10³ transformants per μg of pNZ12 and 2.4 times 10² per μg of pJK108, respectively.     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

Electrotransformation of the stable L-form of Proteus mirabilis

Abstract To improve the transformability of stable protoplast type L-forms of Proteus mirabilis for recombinant plasmid DNA, conditions for efficient electrotransformation were explored. Exposing cells from the exponential phase of growth at a density of 6−8 × 10⁹/ml in electrotransformation buffer having a conductivity of 1.4 mS/cm to a field strength of 6.5 kV/cm for a mean pulse duration time of 1.2 ms reproducibly yielded transformation efficiencies in the order of 5 × 10⁴ transformants per μg of DNA. Compared to the polyethylene glycol method for transformation, electrotransformation appeared to be the method of choice for introduction of plasmid DNA into L-form cells.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

Abstract A method for efficient polyethylene glycol (PEG)-mediated transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA is described. The best conditions found for protoplast regeneration included using 0.45 M sucrose both during the cultivation of the cells and (as an osmotic stabilizer) during their treatment with lysozyme, whereas 0.25 M sodium-succinate was added to the regeneration plates. Under these conditions about 5–10% of input cells regenerated. The highest transformation frequency with plasmid DNA was obtained with a PEG 6000 concentration of 22.5% (w/v). Transforming B. amyloliquefaciens strains with the plasmid pUB110 isolated from B. amyloliquefaciens resulted in 2–4 · 10⁵ transformants/μg DNA, 100–1 000-times as high as with DNA from Bacillus subtilis , suggesting a restriction barrier between the two species. Transformation of B. amyloliquefaciens with plasmids pC194 or pE194 cop -6 gave poor yields and no restriction barrier could be demonstrated for these plasmids. However, by curing pC194 from one of the transformants, a mutant strain compatible to both the plasmids could be isolated, yielding 2–3·10⁴ transformants/μg DNA. Both laboratory and industrial B. amyloliquefaciens strains could be transformed with the procedure.     

Experimental Infection of Nosema sp. Ghose in an Unnatural Host Lesioderma sericorne

ABSTRACT. Various doses of a microsporan parasite, Nosema sp., were fed to third and fourth instar larvae of Lesioderma sericorne that infested different types of stored grains. A spore dose of 3 × 10³ spores/individual resulted in a 39% infection rate, reduction in larval and adult weights, and mean spore concentrations of 1.28 ± 0.2 × 10⁸ spores/larva and 1.1 ± 0.2 × 10⁸ spores/adult. At the above dose, mortality was not well marked (about 35% in larvae and 25% in adults). At 3 × 10⁴ spores/individual, the rate of mortality increases to 80% in larvae and 60% in adults. However, more of the pest population (88% of larvae and 73% of adults) died at a dose of 3 × 10⁵ spores/individual. This dose produced mean spore concentrations of 3.91 ± 0.2 × 10⁸ spores/larva and 2.89 ± 0.2 × 10⁸ spores/adult. Insect death was caused by heavy damage to gut epithelia and fat bodies.     

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Abstract An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis . Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Em^r)] with an efficiency of 3 × 10⁵ transformants per μg plasmid DNA. This high efficiency was obtained by the inclusion in the transformation mixture of liposomes composed of cardiolipin and phosphatidyl choline in a molar ratio of 1 to 6 in the presence of 22.5% polyethylene glycol (PEG). This paper also reports an efficient plasmid transfer method between lactic and streptococci and Bacillus subtilis by means of protoplast fusion. When S. lactis and B. lactis protoplasts undergo fusion mediated by exposure to 37.5% polyethylene glycol, plasmid pGKV21 (3.2 MDa; Em^r) was transfered from one host to the other with a frequency of 10⁻³−10⁻⁵ transformants per regenerating recipient protoplast.     

DIRECT DETECTION OF ESCHERICHIA COLI 0157:H7 IN UNPASTEURIZED APPLE JUICE WITH AN EVANESCENT WAVE BIOSENSOR

An evanescent wave biosensor was used to detect Escherichia coli O157:H7 in unpasteurized apple juice. Light is launched from a 635 nm laser diode into silica or polystyrene optical waveguides, generating an evanescent field which extends from the waveguide surface. Fluorescent molecules within the evanescent field are excited resulting in an emission signal that the biosensor then detects and quantifies. A sandwich immunoassay was performed on the waveguides using cyanine 5 dye-labeled anti-E. coli O157:H7 antibodies for generation of the specific fluorescent signal. The lower limit of detection was between 6.0 × 10² and 6.0 × 10⁴ CFU/mL with silica waveguides and between 3.2 × 10⁴ and 3.2 × 10⁴ CFU/mL using polystyrene waveguides. One-hundred percent correct identification of true positive samples occurred at 6.0 × 10⁴ and 3.2 × 10⁴ CFU/mL for silica and polystyrene waveguides, respectively. Signals from a variety of non-E. coli O157 bacteria, including closely related enterotoxigenic strains of E. coli at concentrations of ˜ 10⁶ CFU/mL, were below the limits of detection. Assays were conducted in near real-time with results obtained within 15 min of sample processing.     

Distribution of Synechococcus spp. determined by immunofluorescent assay

By using two polyclonal antisera against WH 7803 strain (Synechococcus sp.) and WH 5701 strain (Synechococcus bacillaris) it is possible to detect and to enumerate cells of the two cyanobacterial serogroups. The immunofluorescence technique was used to study the distribution of the two serogroups in the estuarine, coastal and upwelling waters of the Mediterranean Sea surrounding Messina. In the estuarine waters of the Alcantara River (Ionian Sea), the WH 7803 serogroup was present at a concentration in the order of 10² cells ml⁻¹ and the WH 5701 serogroup at a concentration of 5·5 × 10² cellsml⁻¹. In the coastal waters of Messina, where urban and industrial wastes are usuallydumped, the concentration of total phycoerythrin- Synechococcus ranged from 1·3 × 10² to 4·1 × 10³ cells ml⁻¹; the WH 7803 serogroup accounted for 50–94% of the totalpopulation in Ionian stations, whereas the WH 5701 serogroup ranged from1·4 × 10¹ to6·7 × 102cells ml⁻¹. In the upwelling area (Straits of Messina) bothserogroups were found. Vertical distribution of two Synechococcus strains had anopposite trend and their concentrations were of the order of 10¹–10²cells ml⁻¹. Theuse of the Scan laser system allows both autofluorescent and labelled organismsto be distinguished in a preparation for optical microscopy. It also allows false-positivecells to be distinguished.     

Relationship between carbon monoxide dehydrogenase and a small plasmid in Pseudomonas carboxydovorans

Abstract Isolation of plasmid DNA followed by plasmid curing was carried out to examine the relationship of plasmid to carbon monoxide dehydrogenase (CO-DH) production in carboxydobacteria. A small plasmid of almost identical size (1.52−1.76 × 10⁶) was present in Pseudomonas carboxydovorans, Azotobacter sp.1, and Azomonas sp.2. Azomonas sp.1 contained two kinds of plasmids (1.5 × 10⁶ and 2.47 × 10⁶). No plasmids were found in Pseudomonas carboxydohydrogena , JC1, and HY1. A plasmid-cured clone of P. carboxydovorans was obtained by growing the cells at 37°C. The cured cell was able to grow CO autotrophically on solid, but not in liquid, medium. CO-DH of the cured cell was active and consisted of three subunits similar to those found in the wild-type enzyme, with the exception that the β subunit of the enzyme was larger than that of the wild-type enzyme. These results suggest that the small plasmids do not carry genes encoding CO-DH but may have gene(s) for processing the β subunit of the enzyme.     

Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids

Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl₂) and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO₄) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5±0.2 × 10⁴ transformants ng⁻¹ plasmid DNA) than with the CaCl₂ (8.1±6.1 × 10⁻¹ transformants ng⁻¹ plasmid) and the 'one-step transformation' protocol (2.5±2.7 transformants ng⁻¹ plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl₂ and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.     

Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue

In vitro and in planta sensitivity of an indirect enzyme-linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10-foldby using a newextraction buffer (gl of : KH₂PO₄, 2; NaHPO₄, 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×10⁷ and 1×10⁸ cells ml⁻¹ with the conventionalextraction buffer phosphate-buffered saline (PBS) and less than 1×10⁶ cells ml⁻¹ when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c.vesicatoria strains, absorbance readings were increased close to three-fold with the lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×10⁷ cfu ml⁻¹ and 1×10⁸ cfu ml⁻¹ with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×10⁵ cfu ml⁻¹ in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure,termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS isthe reacting epitope.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.