Monitoring the fate of genetically engineered bacteria sprayed on the phylloplane of bush beans and grass

Abstract The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn 5 marked Pseudomonas syringae sprayed on the phyllosphere of bush beans was studied in planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5×10⁸ to 3.1×10² cfu g⁻¹ on the phylloplane of grass in the course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial background growth. Southern blot hybridization of Hin dIII digested genomic DNA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacteria the recombinant plasmid was detectable by Southern blot hybridization up to 6×10⁴ cfu g⁻¹ (wet weight). Counts of hybridizing colonies showed that P. syringae established on the phyllosphere of bush beans at between 5×10³ and 4×10⁶ cfu g⁻¹ fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast, Tn5 marked DNA was detected after PCR amplification over the whole period of the experiment.     

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.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans

Abstract A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 × 10² to 2.5 × 10⁴ transformants per 10⁶ viable protoplasts and μg DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

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.     

Production of amylase by the intestinal microflora in cultured freshwater fish

The amylase-producing ability of the intestinal microflora in cultured specimens of ayu, carp, channel catfish, Japanese eel and tilapia was determined. Mean viable counts of aerobes and anaerobes ranged from 1·1×10⁶ to 3·7×10⁸ cfu g⁻¹ and from 1·3×10³ to 1·6×10⁸ cfu g⁻¹, respectively. Aeromonas spp. and Bacteroidaceae were predominant in four to five fish species. Of 206 strains examined, 65 (31·6%) produced ≥0·01 U amylase ml⁻¹. The percentage of producers differed among families and genera of bacteria and fish species. While 56% of the anaerobes produced amylase, only 20% of the aerobes did. More than 50% of Aeromonas , Bacteroidaceae and Clostridium strains produced amylase efficiently while Acinetobacter , coryneforms, Enterobacteriaceae, Moraxella , Plesiomonas and Streptococcus strains did not. High amylase production (≥0·05 U ml⁻¹) was found in 12 strains, 11 from Aeromonas and one Pseudomonas . The percentage of high amylase producers in Japanese eel was lower than the other four fish (2–30%). These results strongly suggest that the amylase produced by the intestinal microflora play an important role in the digestion of starch in freshwater fish to some extent.     

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.     

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.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .     

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.     

CHOLINESTERASE ACTIVITY OF THE MOTOR ENDPLATE IN ISOLATED MUSCLE MEMBRANE

Abstract— The cholinesterase activity of motor endplates in tibialis anterior muscle of rats accounted for about 20 per cent of the total cholinesterase activity of the muscle. In the isolated muscle membrane preparation of rat intercostal muscle, the cholinesterase activity was localized solely in the motor endplate, as shown by cholinesterase staining. The cholinesterase activity of the membrane per unit of nitrogen was 26·9 times that of the muscle homogenate. The membrane (endplate) cholinesterase had an optimal pH of 8, K_m value of 3·1 m m , and was stable at 4° for at least 13 days. Cholinesterase of a motor endplate hydrolysed 2·69 × 10⁸ acetylcholine molecules in 1 msec. Since it is estimated that 10⁸ cholinesterase active sites are present in a motor endplate, the turnover time (time necessary for one enzyme site to hydrolyse one acetylcholine molecule) is calculated to be 372 μ sec, and the turnover number (molecules of acetylcholine hydrolysed by one enzyme site/min) to be 1·61 × 10⁵. From studies with cholinesterase inhibitors, cholinesterase activity was estimated to be due mostly to acetylcholinesterase, and only a minor part to pseudocholinesterase. The muscle membrane preparation seems to be useful for the study of other properties of the motor endplate.     

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 .     

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.     

Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis

Aims:  To elaborate an effective electroporation protocol for large plasmids and wild type strains of Bacillus thuringiensis .
Methods and Results:  The effect of DNA desalting, wall-weakening agency, cell growth conditions, electroporation solutions, and electric fields on electroporation efficiency was evaluated to optimize electroporation conditions for B. thuringiensis . By using this improved method, the greatest efficiency was reached 2 × 10¹⁰ CFU  μ g⁻¹ with pHT304, which is 10⁴ times higher than previously reported. Four large plasmids (29·1, 44·9, 58 and 60 kb) were successfully transferred into the acrystalliferous B. thuringiensis strain BMB171; these results have not been achieved with previous protocols. Three wild type B. thuringiensis strains which could not be transformed previously were also transferred successfully.
Conclusions:  This improved method is more efficient for small plasmids; it is also appropriate for large plasmids and wild type B. thuringiensis strains which were not transformed by previous procedures.
Significance and Impact of the Study:  The present study established an effective electroporation protocol for large plasmids and wild type strains of B. thuringiensis . This method is well suited for the cloning and expression of huge DNA fragments such as gene clusters in B. thuringiensis . It also can be used as a reference method for other Bacillus strains that are refractory to electroporate.     

Sperm allocation in the three-spined stickleback

Male three-spined stickleback Gasterosteus aculeatus have a fixed amount of sperm during the breeding season because spermatogenesis is inhibited at this time. A method was developed to estimate ejaculate size in situ by removing the sperm from the male's nest. The reliability of the method was tested using known numbers of sperm. In their first mating, males ejaculated 11·64 × 10⁶ sperm (median), representing c. 5% of the male's sperm store (median 27·88 × 10⁷ sperm). The amount of sperm in the testes was significantly reduced in males that had mated several times (median 8·09 × 10⁷). Additionally, ejaculate size was smaller in these experienced males (median 8·79 × 10⁵). Heavier and larger fish invested absolutely and relatively more sperm in a mating than did lighter and smaller fish. Ejaculate size did not correlate with the mass of the egg clutch.     

Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

Transient expression of electroporated gene in leaf protoplasts of indica rice and influence of template topology and vector sequences

Electroporation was used for gene delivery and evaluation of various parameters affecting transient expression of a gene for β-glucuronidase ( gus ) in leaf protoplasts of Oryza sativa var. Basmati-370. Transient expression was found to be dependent on voltage, capacitance, amount of plasmid and carrier DNA as well as period of culture. Maximum GUS activity was obtained when a 150 ms pulse at 300 V cm^-1 and 200 μF was applied to the protoplasts (l–2×10⁶ml⁻¹) in an electroporation buffer containing 20 μg of plasmid and 30 μg of calf thymus DNA, assayed 48 h after electroporation. DNA topology did not influence expression of the gene as both linear and supercoiled templates resulted in similar activities, but a 4-fold decrease in expression was observed if the gene was excised, reflecting the positive influence of vector sequences on gene expression.