Generation of a reproducible nutrient-depleted biofilm of Escherichia coli and Burkholderia cepacia

An in vitro method of growing bacteria as a defined nutrient-depleted biofilm is proposed. The medium was defined nutritionally in terms of the quantitative composition and by the total amount of nutrient required to achieve a defined population size. Escherichia coli and Burkholderia cepacia were incubated on a filter support placed on a defined volume of solid medium. The change of biomass of the biofilm population was compared with the change in a planktonic culture. The size of the population in stationary phase was proportional to the concentration of limiting substrate up to 40 μmol cm⁻² glucose for E. coli and up to 2·7 × 10⁻⁹ mol cm⁻² iron for B. cepacia . Escherichia coli growing exponentially had a growth rate of μ = 0·30 h⁻¹ in a biofilm and μ = 0·96 h⁻¹ in planktonic culture. The growth rate, μ, for exponentially growing B. cepacia in a biofilm was 1·12 h⁻¹ and in planktonic culture 0·78 h⁻¹. This method allows the limitation of the size of a biofilm population to a chosen value.     

Electrotransformation of Streptococcus agalactiae with plasmid DNA

Abstract A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli - Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-μl aliquot containing about 5×10⁹ colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×10⁴ cfu μg⁻¹. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae .     

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.     

Influence of the growth rate on the macromolecular composition of Acinetobacter calcoaceticus in carbon-limited chemostat culture

Abstract Infectious phage particles can be formed in vitro when extracts of T1-infected cells are incubated with T1 DNA. The DNA packaging system is based on mixtures of complementing extracts from Escherichia coli sup⁰ cells infected with the amber mutants am 4 (gene 16) or am 10 (gene 13). Gene 16 mutants are defective in the formation of DNA-filled heads but make proheads; gene 13 mutants are defective in prohead formation. Three forms of DNA have been packaged: (1) endogenous concatemeric DNA present in mixtures of am 4 and am 10 mutant extracts; (2) concatemeric DNA; (3) virion DNA both when supplied exogenously to mixtures of am 4 · am 20 and am 10 · am 20 double mutant extracts ( am 20 inhibits T1 DNA synthesis). The reaction requires added ATP, Mg²⁺ and spermidine for optimum efficiency and produces about 1.5 × 10³ pfu/ μ g and about 1 × 10⁴ pfu/ μ g for exogenous concatemeric and virion DNA, respectively.     

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.     

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.     

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.     

Electrotransfection of Propionibacterium freudenreichii TL 110

The first highly efficient protocol is described for the electrotransfection of Propionibacterium freudenreichii with DNA phage. The transfection efficiency is 7 times 10⁵ transfectants per μg of DNA under optimal conditions. Optimized parameters included the field strength (12.5 kV, 200 Ohms, 25 μF), phage DNA concentration (1 μg ml^-1) and cell density (1.5 times 10¹⁰ cells ml^-1). Growth in the presence of glycine and harvesting of cells during the early exponential growth phase increased the transfection efficiency. This electrotransfection protocol is of importance for the genetic improvement of dairy propionibacteria.     

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.     

Ammonium ion affecting tylosin production by Streptomyces fradiae NRRL 2702 in continuous culture

Nitrogen regulation in tylosin production by Streptomyces fradiae NRRL 2702 was studied in chemostat culture using a soluble synthetic medium. The maximum value of specific tylosin formation rate ( q _TYL) was 1·13 mg g⁻¹ h⁻¹ at the specific growth rate (μ) of 0·05 h⁻¹, and q _TYL decreased with increasing levels of the specific growth rate after reaching a rate of 0·1 h⁻¹. The optimum conditions for tylosin formation were that the specific ammonium ion uptake rate ( q _N) and μ were 0·13 mmol g⁻¹ h⁻¹ and 0·05 h⁻¹, respectively. The specific formation rates of threonine dehydratase (TDT) and tylosin were repressed by high levels of specific ammonium ion uptake rate. This study showed the adaptation to chemostat cultures of the nitrogen regulation of tylosin fermentations.     

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.     

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.     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages

The sub-chronic (28–56 days) effects of exposure to low concentrations of cadmium (Cd; 0·05, 0·25, 0·50 and 2·50 μg l⁻¹) shortly following fertilization on embryos, larvae and juvenile rainbow trout Oncorhynchus mykiss were examined. Premature hatching occurred at lower concentrations (0·05 and 0·25 μg l⁻¹ Cd), however, delayed hatching was seen in the 2·50 μg l⁻¹ Cd group, with >90% of hatching occurring on the last day of the hatching period. Larval growth was negatively affected by Cd exposure in a concentration-dependent manner. Larvae exposed to 2·50 μg l⁻¹ Cd were 13·9 ± 0·8% shorter in total length ( L _T) and weighed 22·4 ± 3·5% (mean ± s . e .) less than controls at the end of the exposure period. Plasma sex steroid concentrations (oestradiol in juvenile females and 11-ketotestosterone in juvenile males) were elevated (four- to 10-fold over controls) in exposed fish in both males and females, following 28 days of exposure to 0·05, 0·25 and 0·50 μg l⁻¹ Cd, respectively. These results suggest that environmentally realistic concentrations (in the μg l⁻¹ range) of Cd can affect the development of O. mykiss impacting embryos, larvae and juvenile fish.     

Improved cloning vectors and transformation procedure for Lactococcus lactis

Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1. All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli. By cloning the E. coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E. coli. The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria. One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MGI363 of L. lactis which routinely yielded 1 times 10⁷ to 5 times 10⁷ transformants μg^-1 supercoiled DNA using stored, snap-frozen cells. This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2·5% w.v. Although growth of L. lactis strain MGI363 was inhibited by the use of 0·5 mol 1^-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency. Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength. These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L. lactis.     

Robust experimental design for optimizing the microbial inhibitor test for penicillin detection in milk

Aims:  To use experimental design techniques and a multiple logistic regression model to optimize a microbiological inhibition test with dichotomous response for the detection of Penicillin G in milk.
Methods and Results:  A 2³ × 2² robust experimental design with two replications was used. The effects of three control factors (V: culture medium volume, S: spore concentration of Geobacillus stearothermophilus , I: indicator concentration), two noise factors (Dt: diffusion time, Ip: incubation period) and their interactions were studied. The V, S, Dt, Ip factors and V × S, V × Ip, S × Ip interactions showed significant effects.
Conclusions:  The use of 100  μ l culture medium volume, 2 × 10⁵ spores ml⁻¹, 60 min diffusion time and 3 h incubation period is recommended. In these elaboration conditions, the penicillin detection limit was of 3·9  μ g l⁻¹, similar to the maximum residue limit (MRL). Of the two noise factors studied, the incubation period can be controlled by means of the culture medium volume and spore concentration.
Significance and Impact of the Study:  We were able to optimize bioassays of dichotomous response using an experimental design and logistic regression model for the detection of residues at the level of MRL, aiding in the avoidance of health problems in the consumer.     

A method for long-term measurement of respiration in intertidal fishes during simulated intertidal conditions

Aquatic and aerial respiration of the amphibious fishes Lipophrys pholis and Periophthalmus barbarus were examined using a newly designed flow-through respirometer system. The system allowed long-term measurements of oxygen consumption and carbon dioxide release during periods of aquatic and aerial respiration. The M o ₂ of L. pholis , measured at 15° C, was 2·1 μmol O₂ g^–1 h^–1 during aquatic and 1·99 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the M co₂ were 1.67 and 1.59 μmol O₂ g^–1 h^–1 respectively, giving an aquatic respiratory exchange ratio (RER) of 0·80 and an aerial RER of 0·79. The M o₂ of P. barbarus , measured at 28°C, was 4·05 μmol O₂ g^–1 h^–1 during aquatic and 3·44 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the Mco₂ were 3·29 μmol CO₂ g^–1 h^–1 and 2·63 μmol CO₂ g^–1 h^–1 respectively, giving an aquatic RER of 0·81 and an aerial RER of 0·77. While exposed to air for at least 10 h, both species showed no decrease in metabolic rate or carbon dioxide release. The RER of these fishes equalled their respiratory quotient. After re-immersion an increased oxygen consumption, due to the payment of an oxygen debt, could not be detected.     

Biochemical characterisation of ketoconazole inhibitory action on Aspergillus fumigatus

Abstract The effect of ketoconazole on growth, sterol composition, in vitro sterol biosynthesis and P450-CO complex formation and its interaction with microsomal P450 was determined. On solid medium and in liquid medium ketoconazole inhibited Aspergillus fumigatus growth completely at 5 × 10⁻⁵ M and 50% of the growth at 1.3 × 10⁻⁵ M and 2.1 × 10⁻⁵ M respectively. A close relationship between accumulation of 14α-methyl sterols (eburicol, obtusifoliol and 14α-methyl fecosterol) and depletion of ergosterol with growth arrest was observed in ketoconazole treated cultures. The half inhibitory concentration for in vitro ergosterol biosynthesis and half saturating concentration for type II binding spectrum of ketoconazole were calculated as 73.8 ± 6.3 nM and 0.13 ± 0.04 μM respectively. CO displacement studies revealed inhibition of CO-P450 complex formation by ketoconazole.     

Oxygen consumption by eggs and larvae of striped mullet, Mugil cephalus, in relation to development, salinity and temperature

Oxygen consumption rates during embryonic and the first 38 days of larval development of the striped mullet were measured at 24° C by differential respirometry. Measurements were obtained at the blastula, gastrula and four embryonic stages, and at the yolk-sac, preflexion, flexion and post-flexion larval stages.
Oxygen uptake rates of eggs increased linearly from 0.024 μl O₂ per egg h^-1 (0·323 μl O₂ mg^-1 dry wt h^-1) by blastulae to 0·177 μlO₂ per egg h^-1 (2·516 μlO₂mg ¹dry wth^-1) by embryos prior to hatching. Respiration rates did not vary significantly among four salinities (20,25, 30, 35%₀).
Larval oxygen consumption increased in a curvilinear manner from 0·243 μl O₂ per larva h^-1 shortly after hatching to 18·880 μl O₂ per larva h^-1 on day 38. Oxygen consumption varied in direct proportion to dry weight. Mass-specific oxygen consumption rates of preflexion, flexion, and postflexion larvae did not change with age (10·838 μl O₂ mg ¹dry wt h^-1).
Larval oxygen consumption rates did not vary significantly among salinities 10–35%. Acute temperature increases elicited significant increases in oxygen consumption, these being relatively greater in yolk-sac larvae ( Q₁₀ = 2·75) than in postflexion larvae ( Q₁₀ = 1·40).