Evidence of lethal and sublethal injury in food-borne bacterial pathogens exposed to high-intensity pulsed-plasma gas discharges

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to pulsed-plasma gas discharges (PPGD). METHODS AND RESULTS: The fluorescent redox probe 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used for enumerating actively respiring cells of Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella enterica serovar Typhimurium that were suspended in sterile water at 4 degrees C and exposed to separate PPGD and heat treatments. While there was good agreement between use of respiratory staining (RS) and direct-selective agar plate counting (PC) for enumerating untreated bacteria, there were c. 1 and 3 log-unit differences in surviving cell numbers per millilitre for test organisms subjected to PPGD and heat treatments respectively, when enumerated by these different viability indicators. PPGD-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: Use of this RS method revealed that substantial subpopulations of test bacteria rendered incapable of forming colonies by separate PPGD and heat treatments may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of this RS method offers interesting perspectives on assessing established and novel microbial inactivation methods, and may also provide a better understanding of mechanisms involved in microbial inactivation induced by high-intensity PPGD treatments.     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

Photosynthesis and inorganic carbon transport in isolated asparagus mesophyll cells

The possibility of HCO₃⁻ transport into isolated leaf mesophyll cells of Asparagus sprengeri Regel has been investigated. Measurement of the inorganic carbon pool in these cells over an external pH range 6.2 to 8.0, using the silicone-fluid filtration technique, indicated that the pool was larger than predicted by passive ¹⁴CO₂ distribution, suggesting that HCO₃⁻ as well as CO₂ crosses the plasmalemma. Intracellular pH values, calculated from the distribution of ¹⁴CO₂ between the cells and the medium, were found to be higher (except at pH 8.0) than those previously determined by 5,5-dimethyl[2-¹⁴C]oxazolidine-2,4-dione distribution. It is suggested that the inorganic carbon accumulated above predicted concentrations may be bound to proteins and membranes and thus may not represent inorganic carbon actively accumulated by the cells, inasmuch as in a closed system at constant CO₂ concentration, the photosynthetic rates at pH 7.0 and 8.0 were 5 to 8 times lower than the maximum rate which could be supported by CO₂ arising from the spontaneous dehydration of HCO₃⁻. Furthermore, CO₂ compensation points of the cells in liquid media at 21% O₂ at pH 7.0 and 8.0, and the K_½ CO₂ (CO₂ concentration supporting the half maximal rate of O₂ evolution) at 2% O₂ at pH 7.0 and 8.0 are not consistent with HCO₃⁻ transport. These results indicate that the principal inorganic carbon species crossing the plasmalemma in these cells is CO₂.     

Na-Independent HCO(3) Transport and Accumulation in the Cyanobacterium Synechococcus UTEX 625

The active transport and intracellular accumulation of HCO₃⁻ by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na⁺.Na⁺-dependent HCO₃⁻ accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O₂ evolution and CO₂ fixation. However, when Synechococcus was grown in standing culture, high rates of HCO₃⁻ transport and photosynthesis were observed in the absence of added Na⁺. The internal HCO₃⁻ pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO₃⁻ transport and accumulation in standing culture cells by about 25 to 30% compared with the five- to eightfold enhancement observed with air-grown cells. The ability of standing culture cells to utilize HCO₃⁻ from the medium in the absence of Na⁺ was lost within 16 hours after transfer to air-grown culture and was reacquired during subsequent growth in standing culture. Studies using a mass spectrometer indicated that standing culture cells were also capable of active CO₂ transport involving a high-affinity transport system which was reversibly inhibited by H₂S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO₂ transport system, whereas Na⁺-dependent and Na⁺-independent HCO₃⁻ transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO₃⁻ was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO₂ fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO₃⁻ + CO₂. The pattern of fluorescence quenching observed in response to added HCO₃⁻ and Na⁺ in air-grown and standing culture cells was highly characteristic for Na⁺-dependent and Na⁺-independent HCO₃⁻ accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO₃⁻ transport and the dynamics of intracellular HCO₃⁻ accumulation and dissipation.     

Interspecific hybridisation in rhinoceroses: Confirmation of a Black × White rhinoceros hybrid by karyotype,fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridisation (FISH) and microsatellite analysis

Black and white rhinoceroses are among the most charismatic megaherbivores and have become flagship species for international conservation. They are often subject to intense management that includes being compressed unnaturally in space and density. We present chromosomal and microsatellite evidence to substantiate the first recorded instance of interspecific hybridisation between them. The data suggest that the genetic integrity of the African rhinoceros species probably depends on differences in behavioural and ecological preferences that offer semipermeable reproductive isolation. We caution against the retention of both species in captive and other population situations where disruption of species-specific behaviour patterns may result if there is an unnatural composition in terms of age and sex, and where access to conspecific mates is restricted or absent.     

Characterization of the non-photochemical quenching of chlorophyll fluorescence that occurs during the active accumulation of inorganic carbon in the cyanobacterium Synechococcus PCC 7942

Previous work has shown that the maximum fluorescence yield from PS 2 of Synechococcus PCC 7942 occurs when the cells are at the CO₂ compensation point. The addition of inorganic carbon (C_i), as CO₂ or HCO₃ ^–, causes a lowering of the fluorescence yield due to both photochemical (q_p) and non-photochemical (q_N) quenching. In this paper, we characterize the q_N that is induced by C_i addition to cells grown at high light intensities (500 mol photons m^–2 s^–1). The C_i-induced q_N was considerably greater in these cells than in cells grown at low light intensities (50 mol photons m^–2 s^–1), when assayed at a white light (WL) intensity of 250 mol photons m^–2 s^–1. In high-light grown cells we measured q_N values as high as 70%, while in low-light grown cells the q_N was about 16%. The q_N was relieved when cells regained the CO₂ compensation point, when cells were illuminated by supplemental far-red light (FRL) absorbed mainly by PS 1, or when cells were illuminated with increased WL intensities. These characteristics indicate that the q_N was not a form of energy quenching (q_E). Supplemental FRL illumination caused significant enhancement of photosynthetic O₂ evolution that could be correlated with the changes in q_p and q_N. The increases in q_p induced by C_i addition represent increases in the effective quantum yield of PS 2 due to increased levels of oxidized Q_A. The increase in q_N induced by C_i represents a decrease in PS 2 activity related to decreases in the potential quantum yield. The lack of diagnostic changes in the 77 K fluorescence emission spectrum argue against q_N being related to classical state transitions, in which the decrease in potential quantum yield of PS 2 is due either to a decrease in absorption cross-section or by increased spill-over of excitation energy to PS 1. Both the C_i-induced q_p (t _0.5<0.5 s) and q_N (t _0.51.6 s) were rapidly relieved by the addition of DCMU. The two time constants give further support for two separate quenching mechanisms. We have thus characterized a novel form of q_N in cyanobacteria, not related to state transitions or energy quenching, which is induced by the addition of C_i to cells at the CO₂-compensation point.Abbreviations BTP- 1,3-bis[tris(hydroxymethyl)-methylaminopropane] - Chl- chlorophyll - C_i- inorganic carbon (CO₂+HCO₃ ^–+CO₃ ^2–) - DCMU- 3-(3,4-dichlorophenyl)-, 1-dimethylurea) - F- chlorophyll fluorescence measured at any time in the absence of a saturating flash - F_o- chlorophyll fluorescence with only the weak modulated measuring beam on - F_M'- chlorophyll fluorescence during a saturating flash - F_M- maximum chlorophyll fluorescence, measured in the presence of WL and FRL at the CO₂-compensation point or in the presence of DCMU - F_V- variable fluorescence (= F_M'–F₀) - FRL- supplemental illumination with far red light - MB- modulated measuring beam of the PAM fluorometer - MV- methyl viologen - PAM- pulse amplitude modulation - PFD- incident photon flux density - PS 1, 2- Photosystems 1 and 2 - Q_A- primary electron-accepting plastoquinione of PS 2 - q_N- non-photochemical quenching of chlorophyll fluorescence - q_p- photochemical quenching of chlorophyll fluorescence; rubisco-ribulose bisphosphate carboxylase/oxygenase - SF- saturating flash (600 ms duration) - WL- white light illumination     

Quenching of Chlorophyll a Fluorescence in Response to Na+-Dependent HCO3- Transport-Mediated Accumulation of Inorganic Carbon in the Cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the yield of chlorophyll a fluorescence decreased in response to the transport-mediated accumulation of intracellular inorganic carbon (CO2 + HCO3- + CO32- = dissolved inorganic carbon [DIC]) and subsequently increased to a near-maximum level following photosynthetic depletion of the DIC pool. When DIC accumulation was mediated by the active Na+-dependent HCO3- transport system, the initial rate of fluorescence quenching was found to be highly correlated with the initial rate of H14CO3- transport (r = 0.96), and the extent of fluorescence quenching was correlated with the size of the internal DIC pool (r = 0.99). Na+-dependent HCO3- transport-mediated accumulation of DIC caused fluorescence quenching in either the presence or absence of the CO2 fixation inhibitor glycolaldehyde, indicating that quenching was not due simply to NADP+ reduction. The concentration of Na+ required to attain one-half the maximum rate of H14CO3- transport, at 20 [mu]M external HCO3-, declined from 9 to 1 mM as the external pH increased from 8 to 9.6. A similar pH dependency was observed when fluorescence quenching was used to determine the kinetic constants for HCO3- transport. In cells capable of Na+-dependent HCO3- transport, both the initial rate and extent of fluorescence quenching increased with increasing external HCO3-, saturating at about 150 [mu]M. In contrast Na+-independent HCO3- transport-mediated fluorescence quenching saturated at an HCO3- concentration of about 10 [mu]M. It was concluded that measurement of chlorophyll a fluorescence emission provided a convenient, but indirect, means of following Na+-dependent HCO3- transport and accumulation in Synechococcus.