Dynamics and Distribution of Cyanophages and Their Effect on Marine Synechococcus spp.

Cyanophages infecting marine Synechococcus cells were frequently very abundant and were found in every seawater sample along a transect in the western Gulf of Mexico and during a 28-month period in Aransas Pass, Tex. In Aransas Pass their abundance varied seasonally, with the lowest concentrations coincident with cooler water and lower salinity. Along the transect, viruses infecting Synechococcus strains DC2 and SYN48 ranged in concentration from a few hundred per milliliter at 97 m deep and 83 km offshore to ca. 4 × 10⁵ ml^-1 near the surface at stations within 18 km of the coast. The highest concentrations occurred at the surface, where salinity decreased from ca. 35.5 to 34 ppt and Synechococcus concentrations were greatest. Viruses infecting strains SNC1, SNC2, and 838BG were distributed in a similar manner but were much less abundant (<10 to >5 × 10³ ml^-1). When Synechococcus concentrations exceeded ca. 10³ ml^-1, cyanophage concentrations increased markedly (ca. 10² to > 10⁵ ml^-1), suggesting that a minimum host density was required for efficient viral propagation. Data on the decay rate of viral infectivity d (per day), as a function of solar irradiance I (millimoles of quanta per square meter per second), were used to develop a relationship (d = 0.2610I - 0.00718; r² = 0.69) for conservatively estimating the destruction of infectious viruses in the mixed layer of two offshore stations. Assuming that virus production balances losses and that the burst size is 250, ca. 5 to 7% of Synechococcus cells would be infected daily by viruses. Calculations based on contact rates between Synechococcus cells and infectious viruses produce similar results (5 to 14%). Moreover, balancing estimates of viral production with contact rates for the farthest offshore station required that most Synechococcus cells be susceptible to infection, that most contacts result in infection, and that the burst size be about 324 viruses per lytic event. In contrast, in nearshore waters, where ca. 80% of Synechococcus cells would be contacted daily by infectious cyanophages, only ca. 1% of the contacts would have to result in infection to balance the estimated virus removal rates. These results indicate that cyanophages are an abundant and dynamic component of marine planktonic communities and are probably responsible for lysing a small but significant portion of the Synechococcus population on a daily basis.     

Dynamics and Distribution of Cyanophages and Their Effect on Marine Synechococcus spp

Cyanophages infecting marine Synechococcus cells were frequently very abundant and were found in every seawater sample along a transect in the western Gulf of Mexico and during a 28-month period in Aransas Pass, Tex. In Aransas Pass their abundance varied seasonally, with the lowest concentrations coincident with cooler water and lower salinity. Along the transect, viruses infecting Synechococcus strains DC2 and SYN48 ranged in concentration from a few hundred per milliliter at 97 m deep and 83 km offshore to ca. 4 x 10 ml near the surface at stations within 18 km of the coast. The highest concentrations occurred at the surface, where salinity decreased from ca. 35.5 to 34 ppt and Synechococcus concentrations were greatest. Viruses infecting strains SNC1, SNC2, and 838BG were distributed in a similar manner but were much less abundant (<10 to >5 x 10 ml). When Synechococcus concentrations exceeded ca. 10 ml, cyanophage concentrations increased markedly (ca. 10 to > 10 ml), suggesting that a minimum host density was required for efficient viral propagation. Data on the decay rate of viral infectivity d (per day), as a function of solar irradiance I (millimoles of quanta per square meter per second), were used to develop a relationship (d = 0.2610I - 0.00718; r = 0.69) for conservatively estimating the destruction of infectious viruses in the mixed layer of two offshore stations. Assuming that virus production balances losses and that the burst size is 250, ca. 5 to 7% of Synechococcus cells would be infected daily by viruses. Calculations based on contact rates between Synechococcus cells and infectious viruses produce similar results (5 to 14%). Moreover, balancing estimates of viral production with contact rates for the farthest offshore station required that most Synechococcus cells be susceptible to infection, that most contacts result in infection, and that the burst size be about 324 viruses per lytic event. In contrast, in nearshore waters, where ca. 80% of Synechococcus cells would be contacted daily by infectious cyanophages, only ca. 1% of the contacts would have to result in infection to balance the estimated virus removal rates. These results indicate that cyanophages are an abundant and dynamic component of marine planktonic communities and are probably responsible for lysing a small but significant portion of the Synechococcus population on a daily basis.     

Chemotaxis toward Nitrogenous Compounds by Swimming Strains of Marine Synechococcus spp.

Many of the open-ocean isolates of the marine unicellular cyanobacterium Synechococcus spp. are capable of swimming motility, whereas coastal isolates are nonmotile. Surprisingly, the motile strains do not display phototactic or photophobic responses to light, but they do demonstrate positive chemoresponses to several nitrogenous compounds. The chemotactic responses of Synechococcus strain WH8113 were investigated using blind-well chemotaxis chambers fitted with 3.0-μm-pore-size Nuclepore filters. One well of each chamber contained cells suspended in aged Sargasso Sea water, and the other well contained the potential chemoattractant in seawater. The number of cells that crossed the filter into the attractant-seawater mixture was measured by direct cell counts and compared with values obtained in chambers lacking gradients. Twenty-two compounds were tested, including sugars, amino acids, and simple nitrogenous substrates, at concentrations ranging from 10⁻⁵ to 10⁻¹⁰ M. Strain WH8113 responded positively only to ammonia, nitrate, β-alanine, glycine, and urea. Typically, there was a 1.5- to 2-fold increase in cell concentrations above control levels in chambers containing these compounds, which is comparable to results from similar experiments using enteric and photoheterotrophic bacteria. However, the threshold levels of 10⁻⁹ to 10⁻¹⁰ M found for Synechococcus spp. chemoresponses were lower by several orders of magnitude than those reported for other bacteria and fell within a range that could be ecologically significant in the oligotrophic oceans. The presence of chemotaxis in motile Synechococcus spp. supports the notion that regions of nutrient enrichment, such as the proposed microzones and patches, may play an important role in picoplankton nutrient dynamics.     

Novel Phycoerythrins in Marine Synechococcus spp. : Characterization and Evolutionary and Ecological Implications

Four clones of the marine, unicellular, cyanobacteria Synechococcus spp., were examined for the spectral and biochemical features of their phycoerythrins (PE) and their photosynthetic characteristics. Two spectral types of PE which are distinct from known PEs were found. One PE type possessed absorption maxima at 500 and 545 nm and a fluorescence emission at 560 nm. Upon denaturation in acid-urea, two chromophore absorption maxima were obtained, one corresponding to phycourobilin (A_max 500 nm) and one at 558 nm, ascribed to a phycoerythrobilin-like chromophore. The ratio of phycoerythrobilin-like to phycourobilin chromophores was 4.9:1.3. This PE possessed two subunits of M_rs of 17.0 and 19.5 kD for the α and β subunits, respectively. The other PE possessed a single symmetrical absorption at 551 nm and a fluorescence emission at 570 nm. This phycobiliprotein showed a single chromophore absorption band (A_max 558 nm) and yielded two polypeptides, an α of 17.5 kD and a β subunit of 20.8 kD. Both PEs showed a (α, β)_n structure. The presence of phycoerythrobilin-like chromophores (A_max 558 nm) appears to be diagnostic of this marine cyanobacterial group. The features of these PEs combined with additional biochemical data, suggest a possible evolutionary link between the PE-containing marine Synechococcus group and the red algal chloroplast. When the Synechococcus clones were grown under low light intensity the PE-containing clones showed higher photosynthetic performance, larger photosynthetic units sizes, reaction center I to II ratios near unity, and steeper initial slopes of photosynthesis versus irradiance curves than a non-PE-containing clone. These findings demonstrate the high photosynthetic efficiency of PE-containing clones in low light environments common to middepth neritic and oceanic habitats.     

Photosynthesis and Inorganic Carbon Usage by the Marine Cyanobacterium, Synechococcus sp

The marine cyanobacterium, Synechococcus sp. Nägeli (strain RRIMP N1) changes its affinity for external inorganic carbon used in photosynthesis, depending on the concentration of CO₂ provided during growth. The high affinity for CO₂ + HCO₃⁻ of air-grown cells (K_½ < 80 nanomoles [pH 8.2]) would seem to be the result of the presence of an inducible mechanism which concentrates inorganic carbon (and thus CO₂) within the cells. Silicone-oil centrifugation experiments indicate that the inorganic carbon concentration inside suitably induced cells may be in excess of 1,000-fold greater than that in the surrounding medium, and that this accumulation is dependent upon light energy. The quantum requirements for O₂ evolution appear to be some 2-fold greater for low CO₂-grown cells, compared with high CO₂-grown cells. This presumably is due to the diversion of greater amounts of light energy into inorganic carbon transport in these cells.

A number of experimental approaches to the question of whether CO₂ or HCO₃⁻ is primarily utilized by the inorganic carbon transport system in these cells show that in fact both species are capable of acting as substrate. CO₂, however, is more readily taken up when provided at an equivalent concentration to HCO₃⁻. This discovery suggests that the mechanistic basis for the inorganic carbon concentrating system may not be a simple HCO₃⁻ pump as has been suggested. It is clear, however, that during steady-state photosynthesis in seawater equilibrated with air, HCO₃⁻ uptake into the cell is the primary source of internal inorganic carbon.

     

Chemotaxis toward Nitrogenous Compounds by Swimming Strains of Marine Synechococcus spp

Many of the open-ocean isolates of the marine unicellular cyanobacterium Synechococcus spp. are capable of swimming motility, whereas coastal isolates are nonmotile. Surprisingly, the motile strains do not display phototactic or photophobic responses to light, but they do demonstrate positive chemoresponses to several nitrogenous compounds. The chemotactic responses of Synechococcus strain WH8113 were investigated using blind-well chemotaxis chambers fitted with 3.0-mum-pore-size Nuclepore filters. One well of each chamber contained cells suspended in aged Sargasso Sea water, and the other well contained the potential chemoattractant in seawater. The number of cells that crossed the filter into the attractant-seawater mixture was measured by direct cell counts and compared with values obtained in chambers lacking gradients. Twenty-two compounds were tested, including sugars, amino acids, and simple nitrogenous substrates, at concentrations ranging from 10 to 10 M. Strain WH8113 responded positively only to ammonia, nitrate, beta-alanine, glycine, and urea. Typically, there was a 1.5- to 2-fold increase in cell concentrations above control levels in chambers containing these compounds, which is comparable to results from similar experiments using enteric and photoheterotrophic bacteria. However, the threshold levels of 10 to 10 M found for Synechococcus spp. chemoresponses were lower by several orders of magnitude than those reported for other bacteria and fell within a range that could be ecologically significant in the oligotrophic oceans. The presence of chemotaxis in motile Synechococcus spp. supports the notion that regions of nutrient enrichment, such as the proposed microzones and patches, may play an important role in picoplankton nutrient dynamics.     

Examination of Genetic Relatedness of Marine Synechococcus spp. by Using Restriction Fragment Length Polymorphisms

The relatedness of several marine Synechococcus spp. was estimated by DNA hybridization. Strains isolated from various geographical locations and representing a diversity of DNA base compositions and phycobiliprotein profiles were compared by restriction fragment length polymorphisms for a number of genes. DNAs from two marine red algae and a cryptomonad alga (which exhibit a phycobiliprotein composition similar to that of the marine Synechococcus spp.) and Synechococcus strain PCC6301 (Anacystis nidulans) were also included in the comparison. Strains WH8008, WH8018, and WH7805 were shown to be very similar to one another, as were strains WH7802 and WH7803. Strains WH8110 and WH5701 were clearly unrelated to any of the other strains, and no marine Synechococcus isolate showed any similarity to the freshwater Synechococcus strain PCC6301 or the eucaryotic algae. The method is relatively straightforward and sensitive and uses a variety of basic molecular biology techniques. Its utility in ascertaining the genetic relatedness and diversity of marine Synechococcus spp. and possible extension to field studies are discussed.     

Effect of Temperature and Light on Growth of and Photosynthesis by Synechococcus Isolates Typical of Those Predominating in the Octopus Spring Microbial Mat Community of Yellowstone National Park

Previous molecular analysis of the Octopus Spring cyanobacterial mat revealed numerous genetically distinct 16S rRNA sequences from predominant Synechococcus populations distantly related to the readily cultivated unicellular cyanobacterium Synechococcus lividus. Patterns in genotype distribution relative to temperature and light conditions suggested that the organisms contributing these 16S rRNA sequences may fill distinct ecological niches. To test this hypothesis, Synechococcus isolates were cultivated using a dilution and filtration approach and then shown to be genetically relevant to natural mat populations by comparisons of similarities of 16S rRNA genes and 16S-23S internal transcribed spacer (ITS) regions. Most isolates were identical or nearly identical at both loci to predominant mat genotypes; others showed 1- to 2-nucleotide differences at the 16S rRNA locus and even greater difference in ITS sequences. Isolates with predominant mat genotypes had distinct temperature ranges and optima for growth that were consistent with their distributions in the mat. Isolates with genotypes not previously detected or known to be predominant in the mat exhibited temperature ranges and optima that were not representative of predominant mat populations and also grew more slowly. Temperature effects on photosynthesis did not reflect temperature relations for growth. However, the isolate with the highest temperature optimum and upper limit was capable of performing photosynthesis at a higher temperature than other isolates. Growth rate and photosynthetic responses provided evidence for light acclimation but evidence of, at best, only subtle light adaptation.     

夏季自然高温对桉树光合速率和暗呼吸速率的影响

在湖南夏季的自然高温下,所测7种桉树的日平均净光合速率在12.56～27.85mg·dm     

Effect of Night Temperature on Photosynthesis, Transpiration, and Growth of Spray Carnations

Spray carnation plants were grown for several weeks under an8 h day/16 h night regime at temperatures of approximately 21°C by day and 6, 17, or 30 °C by night. Subsequently,the rates of photosynthesis and transpiration at 20 °C weresimilar. This contrasts with evidence published for some otherspecies. Night temperature had only a slight effect on the plant's growthrate. Leaf area ratios were also similar between treatmentsand for two intervals covering a 5 week period. At the highnight temperature flowers were initiated sooner and there werefewer side shoots per plant than at the lower temperatures. The implications of these results for the optimization of theclimatic environment are discussed briefly, and the resultsare compared with those reported for other species.     

夏季自然高温对桉树光合速率和暗呼吸速率的影响

在湖南夏季的自然高温下,所测7种桉树的日平均净光合速率在12.56～27.85 mg*dm-2*h-1之间,日平均暗呼吸速率在6.06～22.39 mg*dm-2*h-1之间。高温对桉树净光合速率的影响依品种而异。樟脑桉和蓝桉的净光合速率最高峰值出现在35 ℃的气温下,而邓恩桉的净光合速率却受35 ℃气温的显著抑制。桉树暗呼吸速率并不随气温的升高而增大。     

Patterns and regulation of silicon accumulation in Synechococcus spp.

Six clones of the marine cyanobacterium Synechococcus, representing four major clades, were all found to contain significant amounts of silicon in culture. Growth rate was unaffected by silicic acid, Si(OH)₄, concentration between 1 and 120 μM suggesting that Synechococcus lacks an obligate need for silicon (Si). Strains contained two major pools of Si: an aqueous soluble and an aqueous insoluble pool. Soluble pool sizes correspond to estimated intracellular dissolved Si concentrations of 2–24 mM, which would be thermodynamically unstable implying the binding of intracellular soluble Si to organic ligands. The Si content of all clones was inversely related to growth rate and increased with higher [Si(OH)₄] in the growth medium. Accumulation rates showed a unique bilinear response to increasing [Si(OH)₄] from 1 to 500 μM with the rate of Si acquisition increasing abruptly between 80 and 100 μM Si(OH)₄. Although these linear responses imply some form of diffusion‐mediated transport, Si uptake rates at low Si (~1 μM Si) were inhibited by orthophosphate, suggesting a role of phosphate transporters in Si acquisition. Theoretical calculations imply that observed Si acquisition rates are too rapid to be supported by lipid‐solubility diffusion of Si through the plasmalemma; however, facilitated diffusion involving membrane protein channels may suffice. The data are used to construct a working model of the mechanisms governing the Si content and rate of Si acquisition in Synechococcus.     

Growth and H2 production by Synechococcus spp. using organic/inorganic electron donors

Growth with simultaneous photoproduction of H₂ was obtained on various organic and inorganic compounds using axenic cultures of the oxygenic phototrophic bacteria Synechococcus sp. OU 103 and S. cedrorum. Highest H₂ production occurred with resting cells of S. cedrorum on malate (11.8 mmol H₂/vessel), whereas Synechococcus sp. OU 103 preferred sulphide (10.3 mmol H₂/vessel) as electron donor.The authors are with the Microbial Biotechnology Lab. Department of botany, Osmania University, Hyderabad 500 007, India.     

Distribution, Isolation, Host Specificity, and Diversity of Cyanophages Infecting Marine Synechococcus spp. in River Estuaries

The abundance of cyanophages infecting marine Synechococcus spp. increased with increasing salinity in three Georgia coastal rivers. About 80% of the cyanophage isolates were cyanomyoviruses. High cross-infectivity was found among the cyanophages infecting phycoerythrin-containing Synechococcus strains. Cyanophages in the river estuaries were diverse in terms of their morphotypes and genotypes.     

Growth and Photosynthesis of the Cyanobacterium Synechococcus leopoliensis in HCO(3)-Limited Chemostats

Synechococcus leopoliensis was grown in HCO₃⁻-limited chemostats. Growth at 50% the maximum rate occurred when the inorganic carbon concentration was 10 to 15 micromolar (or 5.6 to 8.4 nanomolar CO₂). The O₂ to CO₂ ratios during growth were as high as 192,000 to 1. At growth rates below 80% the maximum rate, essentially all the supplied inorganic carbon was converted to organic carbon, and the cells were carbon limited. Carbon-limited cells used HCO₃⁻ rather than CO₂ for growth. They also exhibited a very high photosynthetic affinity for inorganic carbon in short-term experiments. Cells growing at greater than 80% maximum growth rate, in the presence of high dissolved inorganic carbon, were termed carbon sufficient. These cells had photosynthetic affinities that were about 1000-fold lower than HCO₃⁻-limited cells and also had a reduced capacity for HCO₃⁻ transport. HCO₃⁻-limited cells are reminiscent of the air-grown cells of batch culture studies while the carbon sufficient cells are reminiscent of high-CO₂ grown cells. However, the low affinity cells of the present study were growing at CO₂ concentrations less than air saturation. This suggests that supranormal levels of CO₂ not required to induce the physiological changes usually ascribed to high CO₂ cells.     

Seasonal Variations in Virus-Host Populations in Norwegian Coastal Waters: Focusing on the Cyanophage Community Infecting Marine Synechococcus spp.

Viruses are ubiquitous components of the marine ecosystem. In the current study we investigated seasonal variations in the viral community in Norwegian coastal waters by pulsed-field gel electrophoresis (PFGE). The results demonstrated that the viral community was diverse, displaying dynamic seasonal variation, and that viral populations of 29 different sizes in the range from 26 to 500 kb were present. Virus populations from 260 to 500 kb and dominating autotrophic pico- and nanoeukaryotes showed similar dynamic variations. Using flow cytometry and real-time PCR, we focused in particular on one host-virus system: Synechococcus spp. and cyanophages. The two groups covaried throughout the year and were found in the highest amounts in fall with concentrations of 7.3 × 10⁴ Synechococcus cells ml⁻¹ and 7.2 × 10³ cyanophage ml⁻¹. By using primers targeting the g20 gene in PCRs on DNA extracted from PFGE bands, we demonstrated that cyanophages were found in a genomic size range of 26 to 380 kb. The genetic richness of the cyanophage community, determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified g20 gene fragments, revealed seasonal shifts in the populations, with one community dominating in spring and summer and a different one dominating in fall. Phylogenetic analysis of the sequences originating from PFGE and DGGE bands grouped the sequences into three groups, all with homology to cyanomyoviruses present in cultures. Our results show that the cyanophage community in Norwegian coastal waters is dynamic and genetically diverse and has a surprisingly wide genomic size range.     

Effect of High Temperature on Photosynthesis in Potatoes

The effect of high temperatures on the rate of photosynthesiswas studied in several potato varieties. Temperatures of upto 38 °C did not cause a reduction in the photosynthesisof plants that had been grown at these temperatures for longperiods prior to measurement. Higher temperatures of 40–42°C, or the transfer of plants from daytime temperature regimesof 22 °C to 32 °C, caused a reduction in net photosynthesis.This reduction was found to be essentially mesophyllic in origin.High temperature was found to be associated with a decreasein stomatal resistance, an increase in transpiration, and alarger difference between air and leaf temperatures. Dark respirationrates and compensation points for CO₂ concentration were alsogreater at the high temperatures. It was concluded that thepotato crop can be adopted to grow and have an adequate rateof photosynthesis even at relatively high temperatures. Source-sinkrelationships, which were modified by the later formation oftubers at higher temperatures, did not affect photosynthesisin this study. Varietal differences in resistance to heat stresswere observed, with the clone Cl-884 showing a more efficientcapacity for photosynthesis at temperatures up to 40 °Cthan many commonly grown varieties. High temperature, photosynthesis, potato, Solanum tuberosum L     

Photosynthesis of Marine Macroalgae from Antarctica: Light and Temperature Requirements

The photosynthetic performance of macroalgae isolated in Antarctica was studied in the laboratory. Species investigated were the brown algae Himantothallus grandifolius, Desmarestia anceps, Ascoseira mirabilis, the red algae Palmaria decipiens, Iridaea cordata, Gigartina skottsbergii, and the green algae Enteromorpha bulbosa, Acrosiphonia arcta, Ulothrix subflaccida and U. implexa. Unialgal cultures of the brown and red algae were maintained at 0°C, the green algae were cultivated at 10°C. I_K values were between 18 and 53 μmol m^?2 s^?1 characteristic or low light adapted algae. Only the two Ulothrix species showed higher I_K values between 70 and 74 μmol m^?2 s^?1. Photosynthesis compensated dark respiration at very low photon fluence rates between 1.6 and 10.6 μmol m^?2 s^?1. Values of α were high: between 0.4 and 1.1 μmol O₂ g^?1 FW h^?1 (μmol m^?2 s^?1)^?1 in the brown and red algae and between 2.1 and 4.9 μmol O₂ g^?1 FW h^?1 (μmol m^?2 s^?1)^?1 in the green algal species. At 0°C P_max values of the brown and red algae ranged from 6.8 to 19.1 μmol O₂ g^?1 FW h^?1 and were similarly high or higher than those of comparable Arctic-cold temperate species. Optimum temperatures for photosynthesis were 5 to 10°C in A. mirabilis, 10°C in H. grandifolius, 15°C in G. skottsbergii and 20°C or higher in D. anceps and I. cordata. P: R ratios strongly decreased in most brown and red algae with increasing temperatures due to different Q₁₀ values for photosynthesis (1.4 to 2.5) and dark respiration (2.5 to 4.1). These features indicate considerable physiological adaptation to the prevailing low light conditions and temperatures of Antarctic waters. In this respect the lower depth distribution limits and the northern distribution boundaries of these species partly depend on the physiological properties described here.     

Role of Growth Temperature in Freeze-Thaw Tolerance of Listeria spp.

The food-borne pathogen Listeria monocytogenes can grow in a wide range of temperatures, and several key virulence determinants of the organism are expressed at 37°C but are strongly repressed below 30°C. However, the impact of growth temperature on the ability of the bacteria to tolerate environmental stresses remains poorly understood. In other microorganisms, cold acclimation resulted in enhanced tolerance against freezing and thawing (cryotolerance). In this study, we investigated the impact of growth temperature (4, 25, and 37°C) on the cryotolerance of 14 strains of L. monocytogenes from outbreaks and from food processing plant environments and four strains of nonpathogenic Listeria spp. (L. welshimeri and L. innocua). After growth at different temperatures, cells were frozen at −20°C, and repeated freeze-thaw cycles were applied every 24 h. Pronounced cryotolerance was exhibited by cells grown at 37°C, with a <1-log decrease after 18 cycles of freezing and thawing. In contrast, freeze-thaw tolerance was significantly reduced (P < 0.05) when bacteria were grown at either 4 or 25°C, with log decreases after 18 freeze-thaw cycles ranging from 2 to >4, depending on the strain. These findings suggest that growth at 37°C, a temperature required for expression of virulence determinants of L. monocytogenes, is also required for protection against freeze-thaw stress. The negative impact of growth at low temperature on freeze-thaw stress was unexpected and has not been reported before with this or other psychrotrophic microorganisms.Listeria monocytogenes remains a leading cause of deaths due to food-borne illness in the United States and other industrialized nations. Neonates, pregnant women, and immunocompromised people are at high risk for infection (15, 28, 37). Outbreaks of listeriosis tend to involve a relatively small number of closely related strains (“epidemic clones”), primarily of serotype 4b. Several major outbreaks have been attributed to epidemic clone I (ECI) and epidemic clone II (ECII), both of serotype 4b (5, 21).Unlike most other human food-borne pathogens, L. monocytogenes grows over a wide temperature range (1 to 45°C), with optimal growth at approximately 37°C (33). It has been known for some time that expression of several key virulence genes, including hly, encoding the hemolysin listeriolysin O and actA, encoding a protein that mediates actin polymerization required for intracellular pathogenesis, is optimal at 37°C but severely repressed at temperatures below 30°C (22). In contrast, flagellin, motility, and chemotaxis genes are repressed at 37°C but optimally expressed at temperatures below 25°C (9, 10).For an organism such as L. monocytogenes, which is commonly found in the environment but can also colonize and infect warm-blooded animals, temperature is likely to serve as a major signal differentiating environmental from vertebrate host-associated habitats (19). However, with the exception of the thermoregulated phenotypes described above (virulence factor production, motility, and chemotaxis), the impact of growth at different temperatures on specific responses and adaptations of the pathogen remains poorly understood.L. monocytogenes may be exposed to freezing as well as thawing in the course of its existence in natural environments (e.g., soils and water in temperate or cold regions), as well as during the storage and preservation of foods. The organism has been repeatedly isolated from frozen foods (e.g., ice cream) (7). After freezing (−18°C) in laboratory media or in foods and a single thawing cycle, survival depended on strain, freezing medium, and the presence of glycerol as cryoprotectant (13, 14). The freezing and thawing of bacteria grown at 30°C, in combination with essential oil, has been explored as one means to reduce the pathogen in foods (8). The possible role of the general stress sigma factor (sigma B) in survival of bacteria grown at 30°C and exposed to repeated freezing and thawing was also investigated (43). However, there is a surprising dearth of information on the possible impact of growth temperature on tolerance of L. monocytogenes to repeated freezing and thawing (cryotolerance).Studies with another gram-positive psychrotrophic bacterium (Exiguobacterium sibiricum and other Exiguobacterium spp.) revealed that growth in the cold (4°C), or growth on solid media regardless of temperature, resulted in increased tolerance of the bacteria against repeated freezing and thawing (40). It is not known whether low temperature or surface-associated growth may exert similar impacts on the cryotolerance of L. monocytogenes. The objective of the present study was to investigate the impact of growth temperature (4, 25, and 37°C) and of planktonic versus agar growth of L. monocytogenes on protection of the bacteria against repeated freezing and thawing.     

In Situ Hybridization of Prochlorococcus and Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.