Listeria monocytogenes Scott A: cell surface charge, hydrophobicity, and electron donor and acceptor characteristics under different environmental growth conditions

We determined the variations in the surface physicochemical properties of Listeria monocytogenes Scott A cells that occurred under various environmental conditions. The surface charges, the hydrophobicities, and the electron donor and acceptor characteristics of L. monocytogenes Scott A cells were compared after the organism was grown in different growth media and at different temperatures; to do this, we used microelectrophoresis and the microbial adhesion to solvents method. Supplementing the growth media with glucose or lactic acid affected the electrical, hydrophobic, and electron donor and acceptor properties of the cells, whereas the growth temperature (37, 20, 15, or 8 degrees C) primarily affected the electrical and electron donor and acceptor properties. The nonlinear effects of the growth temperature on the physicochemical properties of the cells were similar for cells cultivated in two different growth media, but bacteria cultivated in Trypticase soy broth supplemented with 6 g of yeast extract per liter (TSYE) were slightly more hydrophobic than cells cultivated in brain heart infusion medium (P < 0.05). Adhesion experiments conducted with L. monocytogenes Scott A cells cultivated in TSYE at 37, 20, 15, and 8 degrees C and then suspended in a sodium chloride solution (1.5 x 10(-1) or 1.5 x 10(-3) M NaCl) confirmed that the cell surface charge and the electron donor and acceptor properties of the cells had an influence on their attachment to stainless steel.     

Soft cheese supplemented with black cumin oil: Impact on food borne pathogens and quality during storage

Black cumin seed oil (BCSO) was tested for its inhibitory effect against some pathogenic bacteria (Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Listeria monocytogenes Scott A and Salmonella enteritidis PT4) in Domiati cheese during cold storage. Physical, chemical and sensorial changes in cheese during storage were recorded. Pasteurized milk was inoculated before renneting with a mixed culture of bacteria at ca. 4 log CFU mL⁻¹. In vitro and in situ supplementation with BCSO showed antimicrobial impact on the growth of S. aureus, E. coli, L. monocytogenes and S. enteritidis inoculated into media and cheese samples. Supplementing of cheese with BCSO (0.1% or 0.2%, w/w) significantly reduced the counts of the inoculated pathogens by ca. 1.3 log and 1.5 log CFU g⁻¹ after 21 days of storage. In addition, BCSO controlled the development of titratable acidity, limited the changes in ripening indices, flavor components and kept considerable physicochemical and sensorial properties of cheese.     

Growth of a Dehalococcoides-Like Microorganism on Vinyl Chloride and cis-Dichloroethene as Electron Acceptors as Determined by Competitive PCR

A competitive PCR (cPCR) assay targeting 16S ribosomal DNA was developed to enumerate growth of a Dehalococcoides-like microorganism, bacterium VS, from a mixed culture catalyzing the reductive dehalogenation of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), with hydrogen being used as an electron donor. The growth of bacterium VS was found to be coupled to the dehalogenation of VC and cDCE, suggesting unique metabolic capabilities. The average growth yield was (5.2 ± 1.5) × 10⁸ copies of the 16S rRNA gene/μmol of Cl⁻ (number of samples, 10), with VC being used as the electron acceptor and hydrogen as the electron donor. The maximum VC utilization rate () was determined to be 7.8 × 10⁻¹⁰ μmol of Cl⁻ (copy⁻¹ day⁻¹), indicating a maximum growth rate of 0.4 day⁻¹. These average growth yield and values agree well with values found previously for dechlorinating cultures. Decay coefficients were determined with growth (0.05 day⁻¹) and no-growth (0.09 day⁻¹) conditions. An important limitation of this cPCR assay was its inability to discriminate between active and inactive cells. This is an essential consideration for kinetic studies.     

Listeria monocytogenes LO28: Surface Physicochemical Properties and Ability To Form Biofilms at Different Temperatures and Growth Phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20°C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37°C. At 8°C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Kinetics of Perchlorate- and Chlorate-Respiring Bacteria

Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated “Dechlorosoma” sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h⁻¹, respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h⁻¹, respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h⁻¹, respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h⁻¹, respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h⁻¹) and exceeded that of strain KJ on perchlorate (0.14 h⁻¹). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 ± 0.07 [n = 7]), chlorate (0.44 ± 0.05 [n = 7]), and perchlorate (0.50 ± 0.08 [n = 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.     

Kinetics of Denitrifying Growth by Fast-Growing Cowpea Rhizobia

Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO₃⁻, NO₂⁻, and N₂O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (K_s) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: K_s, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq⁻¹; μ_max, 0.41 and 0.33 h⁻¹. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent K_s values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the K_s for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O₂ is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μ_max, 0.41 h⁻¹) when A26 was grown with either O₂ or NO₃⁻ as an electron acceptor and were only slightly reduced when A28 was grown with NO₃⁻ (0.25 h⁻¹) as opposed to O₂ (0.33 h⁻¹).     

Cold Shock Induction of Thermal Sensitivity in Listeria monocytogenes

Cold shock at 0 to 15°C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60°C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8°C for controls and 7.7°C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28°C followed by heating at 60°C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D₆₀ values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.     

Expression of ActA, Ami, InlB, and Listeriolysin O in Listeria monocytogenes of Human and Food Origin

Expression of proteins involved in the adhesion of Listeria monocytogenes to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of L. monocytogenes strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90; P = 1 × 10⁻⁴). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89; P = 1 × 10⁻²) and central nervous system infections were associated with group ActA2 strains (OR = 3.04; P = 1 × 10⁻³) and group ActA3 strains (OR = 3.91; P = 1 × 10⁻³).     

Surface of Lactic Acid Bacteria: Relationships between Chemical Composition and Physicochemical Properties

The surface chemical composition and physicochemical properties (hydrophobicity and zeta potential) of two lactic acid bacteria, Lactococcus lactis subsp. lactis bv. diacetilactis and Lactobacillus helveticus, have been investigated using cells harvested in exponential or stationary growth phase. The surface composition determined by X-ray photoelectron spectroscopy (XPS) was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbonlike compounds. The concentration of the last was always below 15% (wt/wt), which is related to the hydrophilic character revealed by water contact angles of less than 30°. The surfaces of L. lactis cells had a polysaccharide concentration about twice that of proteins. The S-layer of L. helveticus was either interrupted or crossed by polysaccharide-rich compounds; the concentration of the latter was higher in the stationary growth phase than in the exponential growth phase. Further progress was made in the interpretation of XPS data in terms of chemical functions by showing that the oxygen component at 531.2 eV contains a contribution of phosphate in addition to the main contribution of the peptide link. The isoelectric points were around 2 and 3, and the electrophoretic mobilities above pH 5 (ionic strength, 1 mM) were about −3.0 × 10⁻⁸ and −0.6 × 10⁻⁸ m² s⁻¹ V⁻¹ for L. lactis and L. helveticus, respectively. The electrokinetic properties of the latter reveal the influence of carboxyl groups, while the difference between the two strains is related to a difference between N/P surface concentration ratios, reflecting the relative exposure of proteins and phosphate groups at the surface.     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H2-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H₂-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 ± 0.19 mol%), a phospholipid pattern of type II, MK-9(H₄) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t_d = 8 h), H₂ plus CO₂ (t_d = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H₂, nickel was not necessary. The optimum growth temperature was 65°C; no growth was observed below 40°C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70°C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K_m was 88 μl of CO liter⁻¹ and V_max was 20.2 μl of CO h⁻¹ mg of protein⁻¹. The threshold value of S. thermoautotrophicus of 0.2 μl of CO liter⁻¹ was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 μmol min⁻¹ mg of protein⁻¹. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.     

Enrichment of Denitrifying Methane-Oxidizing Microorganisms Using Up-Flow Continuous Reactors and Batch Cultures

Denitrifying anaerobic methane oxidizing (DAMO) microorganisms were enriched from paddy field soils using continuous-flow and batch cultures fed with nitrate or nitrite as a sole electron acceptor. After several months of cultivation, the continuous-flow cultures using nitrite showed remarkable simultaneous methane oxidation and nitrite reduction and DAMO bacteria belonging to phylum NC10 were enriched. A maximum volumetric nitrite consumption rate of 70.4±3.4 mg-N·L⁻¹·day⁻¹ was achieved with very short hydraulic retention time of 2.1 hour. In the culture, about 68% of total microbial cells were bacteria and no archaeal cells were detected by fluorescence in situ hybridization. In the nitrate-fed continuous-flow cultures, 58% of total microbial cells were bacteria while archaeal cells accounted for 7% of total cell numbers. Phylogenetic analysis of pmoA gene sequence showed that enriched DAMO bacteria in the continuous-flow cultivation had over 98% sequence similarity to DAMO bacteria in the inoculum. In contrast, for batch culture, the enriched pmoA gene sequences had 89–91% sequence similarity to DAMO bacteria in the inoculum. These results indicate that electron acceptor and cultivation method strongly affect the microbial community structures of DAMO consortia.     

Tetrathionate-Forming Thiosulfate Dehydrogenase from the Acidophilic,Chemolithoautotrophic Bacterium Acidithiobacillus ferrooxidans

Thiosulfate dehydrogenase is known to play a significant role in thiosulfate oxidation in the acidophilic, obligately chemolithoautotroph, Acidithiobacillus ferrooxidans. Enzyme activity measured using ferricyanide as the electron acceptor was detected in cell extracts of A. ferrooxidans ATCC 23270 grown on tetrathionate or sulfur, but no activity was detected in ferrous iron-grown cells. The enzyme was enriched 63-fold from cell extracts of tetrathionate-grown cells. Maximum enzyme activity (13.8 U mg⁻¹) was observed at pH 2.5 and 70°C. The end product of the enzyme reaction was tetrathionate. The enzyme reduced neither ubiquinone nor horse heart cytochrome c, which serves as an electron acceptor. A major protein with a molecular mass of ∼25 kDa was detected in the partially purified preparation. Heme was not detected in the preparation, according to the results of spectroscopic analysis and heme staining. The open reading frame of AFE_0042 was identified by BLAST by using the N-terminal amino acid sequence of the protein. The gene was found within a region that was previously noted for sulfur metabolism-related gene clustering. The recombinant protein produced in Escherichia coli had a molecular mass of ∼25 kDa and showed thiosulfate dehydrogenase activity, with maximum enzyme activity (6.5 U mg⁻¹) observed at pH 2.5 and 50°C.     

Structure-Activity Relationships in Microbial Transformation of Phenols

The second-order rate constants for the microbial transformation of a series of phenols were correlated with the physicochemical properties of the phenols. The compounds studied were phenol, p-methylphenol, p-chlorophenol, p-bromophenol, p-cyanophenol, p-nitrophenol, p-acetylphenol, and p-methoxyphenol. Phenol-grown cells of Pseudomonas putida U transformed these compounds. Microbial transformation rate constants ranged from (1.5 ± 0.99) × 10⁻¹⁴ liter · organism⁻¹ · h⁻¹ for p-cyanophenol to (7.0 ± 1.3) × 10⁻¹² liter · organism⁻¹ · h⁻¹ for phenol. Linear regression analyses of rate constants and electronic, steric, and hydrophobic parameters showed that van der Waal's radii gave the best coefficient of determination (r² = 0.956). Products identified by thin-layer chromatography and liquid chromatography indicated that the phenols were microbially oxidized to the corresponding catechols.     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Shoot culture of Bacopa monnieri: standardization of explant,vessels and bioreactor for growth and antioxidant capacity

Standardization of biomass production in different vessels and bioreactor using explants and media for growth, total phenolic content and antioxidant capacity of shoot culture of Bacopa monnieri is described. Maximum number of shoots per explant, higher explants response irrespective of the type of explants, and higher shoot length was obtained on MS medium containing BAP (2.5 mg l⁻¹) and IAA (0.01 mg l⁻¹) with 3 % sucrose. This medium was selected by varying BAP concentration and recorded optimal for shoot culture on gelled medium. The condition of 0.5 cm explant size and 20 explant/40 ml (1 explant/2 ml) was optimal for high explant response, number of shoots per explant regenerated and shoots length. Among the different vessels used, maximum growth index was achieved in Growtek bioreactor (10.0) followed by magenta box (9.16), industrial glass jar (7.7) and conical flask (7.2). The cultures grown in conical flask (100 ml) were used as control. The total phenolic content and antioxidant capacity of in vitro grown plants was higher to that recorded for in vivo material. Among in vitro regenerated plants, the activity was maximal in the tissues grown in 250 ml conical flask. The most critical function for vessels is to support the optimum profusion (growing area for maximum growth) of shoots and for B. monnieri, Growtek bioreactor supported 1980 shoots l⁻¹ medium as compared to control (938 shoots l⁻¹). Growtek bioreactor was considered effective system to produce B. monnieri biomass in culture without loss of antioxidant properties.     

Selection and Identification of a Listeria monocytogenes Target Strain for Pulsed Electric Field Process Optimization

Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm. When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23°C for 144 μs, inactivation ranged from 0.7 to 3.7 log₁₀ CFU/ml. Inactivation by 72-μs PEF treatments at 37°C ranged from 0.3 to 2.5 log₁₀ CFU/ml. L. monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains. The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2). Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A. Use of L. monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L. monocytogenes. The nine L. monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques. These strains were better differentiated with PFGE than with AP-PCR. The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.     

Cloning, Expression, and Purification of Choline Dehydrogenase from the Moderate Halophile Halomonas elongata

Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate. Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures. To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro. In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase. The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose. The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 μmol of O₂ min⁻¹ mg⁻¹ mM⁻¹ at pH 7 and 25°C, respectively. With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent V_max values for choline and betaine-aldehyde were 10.9 and 5.7 μmol of O₂ min⁻¹ mg⁻¹, respectively. These V_max values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor. Altogether, the kinetic data are consistent with the conclusion that H. elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m⁻² s⁻¹) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m⁻² s⁻¹) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.