Relationships between Biovolume and Biomass of Naturally Derived Marine Bacterioplankton

Microscopic estimation of bacterial biomass requires determination of both biovolume and biovolume-to-biomass conversion. Both steps have uncertainty when applied to the very small bacteria typically found in natural seawater. In the present study, natural bacterioplankton assemblages were freshly collected, passed through 0.6-μm-pore-size Nuclepore filters to remove larger particulate materials, and diluted for growth in 0.22-μm-pore-size Millipore filter-sterilized unenriched seawater. This provided cells comparable in size and morphology to those in natural seawater, but the cultures were free of the interfering particulate detritus naturally present. Cells were collected on glass-fiber GF/F filters, and biovolumes were corrected for cells passing these filters; C and N were measured with a CHN analyzer. Our criteria for size measurement by epifluorescence photomicrography were confirmed with fluorescent microspheres of known diameters. Surprisingly, in six cultures with average per-cell biovolumes ranging from 0.036 to 0.073 μm³, the average per-cell carbon biomass was relatively constant at 20 ± 0.08 fg of C (mean ± standard error of the mean). The biovolume-to-biomass conversion factor averaged 0.38 ± 0.05 g of C cm⁻³, which is about three times higher than the value previously estimated from Escherichia coli, and decreased with increasing cell volume. The C:N ratio was 3.7 ± 0.2. We conclude that natural marine bacterial biomass and production may be higher than was previously thought and that variations in bacterial size may not reflect variations in biomass per cell.     

Annual Bacterioplankton Biomasses and Productivities in a Temperate West Coast Canadian Fjord

Bacterioplankton numbers, biomasses, and productivities, as well as chlorophyll a concentrations and phytoplankton productivities, were assayed from 1 March 1984 to 12 August 1985 through a 250-m-deep seawater column in Howe Sound, a temperate fjord-sound on the southern coast of British Columbia, Canada. Primary production during this 18-month period was 845 g of C m⁻². Bacterial production was assayed over this same period as 193 g of C m⁻² (thymidine incorporation) and 77 g of C m⁻² (frequency of dividing cells). Bacterial productivities per cubic meter were usually greater in the euphotic zone than in deeper aphotic water, but when integrated through the water column, approximately half of the bacterial production occurred in the deeper aphotic portion. Bacterial production occurred throughout the year, although at reduced rates in late fall and early winter; primary production almost ceased during late fall and early winter. Because of this heterotrophic bacterioplankton production was a very large portion of the microbial (bacterial plus phyto-plankton) production at this time. In mid-summer bacterial production was a small proportion of the microbial production. Because of this asynchrony in peaks and troughs of bacterial and phytoplankton production through the year, data comparison is best done over an annual cycle. On this basis the bacterial production in the Howe Sound water column was between 23 and 9% of the phytoplankton production when a bacterial C to biovolume ratio of 0.107 pg of C μm⁻³ was assumed; the corresponding values were 64 and 29% when a ratio of 0.300 pg of bacterial C μm⁻³ was assumed.     

Assessing Phytoplankton and Bacterioplankton Production During Early Spring in Lake Erken, Sweden

The spring development of both phytoplankton and bacterioplankton was investigated between 18 April and 7 May 1983 in mesotrophic Lake Erken, Sweden. By using the lake as a batch culture, our aim was to estimate, via different methods, the production of phytoplankton and bacterioplankton in the lake and to compare these production estimates with the actual increase in phytoplankton and bacterioplankton biomass. The average water temperature was 3.5°C. Of the phytoplankton biomass, >90% was the diatom Stephanodiscus hantzchii var. pusillus, by the peak of the bloom. The ¹⁴C and O₂ methods of estimating primary production gave equivalent results (r = 0.999) with a photosynthetic quotient of 1.63. The theoretical photosynthetic quotient predicted from the C/NO₃ N assimilation ratio was 1.57. The total integrated incorporation of [¹⁴C]bicarbonate into particulate material (>1 μm) was similar to the increase in phytoplankton carbon determined from cell counts. Bacterioplankton increased from 0.5 × 10⁹ to 1.52 × 10⁹ cells liter⁻¹ (~0.5 μg of C liter⁻¹ day⁻¹). Estimates of bacterioplankton production from rates of [³H]thymidine incorporation were ca. 1.2 to 1.7 μg of C liter⁻¹ day⁻¹. Bacterial respiration, measured by a high-precision Winkler technique, was estimated as 4.8 μg of C liter⁻¹ day⁻¹, indicating a bacterial growth yield of 25%. The bulk of the bacterioplankton production was accounted for by algal extracellular products. Gross bacterioplankton production (production plus respiration) was 20% of gross primary production, per square meter of surface area. We found no indication that bacterioplankton production was underestimated by the [³H]thymidine incorporation method.     

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW of Escherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V^0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm³ (n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm³) to 1,177 fg (V = 3.5 μm³). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to V of individual cells varied from 466 fg of C μm⁻³ for Vs of 0.001 to 0.01 μm³ to 397 fg of C μm⁻³ (0.01 to 0.1 μm³) and 288 fg of C μm⁻³ (0.1 to 1 μm³). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm⁻³ (0.1 to 1 μm³) and 211 fg of C μm⁻³ (1 to 4 μm³), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.     

Automatic Determination of Bacterioplankton Biomass by Image Analysis

Image analysis was applied to epifluorescense microscopy of acridine orange-stained plankton samples. A program was developed for discrimination and binary segmentation of digitized video images, taken by an ultrasensitive video camera mounted on the microscope. Cell volumes were estimated from area and perimeter of the objects in the binary image. The program was tested on fluorescent latex beads of known diameters. Biovolumes measured by image analysis were compared with directly determined carbon biomasses in batch cultures of estuarine and freshwater bacterioplankton. This calibration revealed an empirical conversion factor from biovolume to biomass of 0.35 pg of C μm⁻³ (± 0.03 95% confidence limit). The deviation of this value from the normally used conversion factors of 0.086 to 0.121 pg of C μm⁻³ is discussed. The described system was capable of measuring 250 cells within 10 min, providing estimates of cell number, mean cell volume, and biovolume with a precision of 5%.     

Estimating the Growth Rate of Slowly Growing Marine Bacteria from RNA Content

In past studies of enteric bacteria such as Escherichia coli, various measures of cellular RNA content have been shown to be strongly correlated with growth rate. We examined this correlation for four marine bacterial isolates. Isolates were grown in chemostats at four or five dilution rates, yielding growth rates that spanned the range typically determined for marine bacterial communities in nature (μ = 0.01 to 0.25 h^-1). All measures of RNA content (RNA cell^-1, RNA:biovolume ratio, RNA:DNA ratio, RNA:DNA:biovolume ratio) were significantly different among isolates. Normalizing RNA content to cell volume substantially reduced, but did not eliminate, these differences. On average, the correlation between μ and the RNA:DNA ratio accounted for 94% of variance when isolates were considered individually. For data pooled across isolates (analogous to an average measurement for a community), the ratio of RNA:DNA μm^-3 (cell volume) accounted for nearly half of variance in μ (r² = 0.47). The maximum RNA:DNA ratio for each isolate was extrapolated from regressions. The regression of (RNA:DNA)/(RNA:DNA)_max on μ was highly significant (r² = 0.76 for data pooled across four isolates) and virtually identical for three of the four isolates, perhaps reflecting an underlying common relationship between RNA content and growth rate. The dissimilar isolate was the only one derived from sediment. Cellular RNA content is likely to be a useful predictor of growth rate for slowly growing marine bacteria but in practice may be most successful when applied at the level of individual species.     

Extracellular Polymeric Substance Architecture Influences Natural Genetic Transformation of Acinetobacter baylyi in Biofilms

Genetic exchange by natural transformation is an important mechanism of horizontal gene transfer in biofilms. Thirty-two biofilm metrics were quantified in a heavily encapsulated Acinetobacter baylyi strain and a miniencapsulated mutant strain, accounting for cellular architecture, extracellular polymeric substances (EPS) architecture, and their combined biofilm architecture. In general, transformation location, abundance, and frequency were more closely correlated to EPS architecture than to cellular or combined architecture. Transformation frequency and transformant location had the greatest correlation with the EPS metric surface area-to-biovolume ratio. Transformation frequency peaked when EPS surface area-to-biovolume ratio was greater than 3 μm²/μm³ and less than 5 μm²/μm³. Transformant location shifted toward the biofilm-bulk fluid interface as the EPS surface area-to-biovolume ratio increased. Transformant biovolume was most closely correlated with EPS biovolume and peaked when transformation occurred in close proximity to the substratum. This study demonstrates that biofilm architecture influences A. baylyi transformation frequency and transformant location and abundance. The major role of EPS may be to facilitate the binding and stabilization of plasmid DNA for cellular uptake.     

A study of lipid bilayer membrane stability using precise measurements of specific capacitance

A method is described for measuring the specific capacitance (C_m) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10^-2 M KCl show that C_m depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), C_m increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm² with a time constant of ~15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. C_m of final-state membranes depends upon applied voltage (V_a) and obeys the equation C_m = C₀ + βV_a² where V_a V_DC + V^rms_AC. C₀ and β depend upon temperature; C₀ decreases linearly with temperature while β increases linearly. At 20°C, C₀ = 0.559 ±0.01 (SD) μF/cm² and β = 0.0123 ±0.0036 (SD) (μF/cm²)/(mv²) and at 34°C, C₀ = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in C_m are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves.     

Change in Size of Chromatium minus Cells in Relation to Growth Rate, Sulfur Content, and Photosynthetic Activity: A Comparison of Pure Cultures and Field Populations

The size frequency distribution of planktonic cells of purple sulfur phototrophic bacteria was measured at several depths in a bacterial layer of Lake Cisó (Spain). The bacterioplankton was dominated by Chromatium minus (87 to 94% of the total biomass). The largest cells of C. minus were found in the top part of the bacterial layer. In addition, the in situ and potential specific photosynthetic activity (CO₂ fixation and acetate uptake) and specific pigment content were measured in relation to several key environmental parameters that determine the activity of cells. Potential growth rates were estimated from production rates and biomass. A maximal specific growth rate of 0.074 h⁻¹ was found for the top part of the bacterial layer. Photosynthesis versus light and versus sulfide curves among field samples indicated that light was the main limiting factor controlling the activity of C. minus in Lake Cisó. The specific bacteriochlorophyll a content was very high in all samples (0.27 to 0.36 μg μg of C⁻¹). Results of laboratory experiments performed with pure cultures indicated that the average cell volume changes from 5.9 to 20.0 μm³ and that differences in growth rate, breakdown, or synthesis of sulfur and glycogen and degradation of the photosynthetic apparatus are the main factors accounting for the observed changes in cell volume across the bacterial layer.     

Cellular Microcystin Content in N-Limited Microcystis aeruginosa Can Be Predicted from Growth Rate

Cell quotas of microcystin (Q_MCYST; femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q_MCYST and specific growth rate (μ), from which we propose a generalized model that enables Q_MCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q_MCYST from μ, μ_max (maximum specific growth rate), Q_MCYSTmax (maximum cell quota), and Q_MCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict a Q_MCYSTmax of 0.129 fmol cell⁻¹ at μ_max and a Q_MCYSTmin of 0.050 fmol cell⁻¹ at μ = 0. Net MCYST production rate (R_MCYST) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell⁻¹ day⁻¹ at μ_max. MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST/protein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chl a ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.     

Carbon- and Nitrogen-to-Volume Ratios of Bacterioplankton Grown under Different Nutritional Conditions

Carbon- and nitrogen-to-volume (C/V and N/V) ratios were determined for freshwater bacterial assemblages grown in lake water filtrate or in water enriched with nutrients (aqueous extract of lake seston, glucose, arginine, phosphate, or ammonium). Biovolume was measured by epifluorescence microphotography, and carbon and nitrogen biomasses were measured with a CHN analyzer. Despite large variations of nutritional conditions (i.e., the composition and concentration of the dissolved organic carbon) and different mean cell sizes of the bacterial assemblage (0.17 to 1.8 μm³ per cell), the C/V, N/V, and carbon-to-nitrogen weight ratios varied little (C/V ratio, 0.14 pg of C per μm³ [standard deviation, 0.057; n = 15]; N/V ratio, 0.027 pg of N per μm³ [standard deviation; 0.011, n = 15]; carbon-to-nitrogen weight ratio, 5.6 [standard deviation, 2.2, n = 15]). An average C/V ratio of 0.12 pg of C per μm³ that was derived from natural and cultured bacterial assemblages is proposed as an appropriate conversion factor for estimation of the biomass of freshwater bacteria.     

Response of Bacterioplankton Communities to Cadmium Exposure in Coastal Water Microcosms with High Temporal Variability

Multiple anthropogenic disturbances to bacterial diversity have been investigated in coastal ecosystems, in which temporal variability in the bacterioplankton community has been considered a ubiquitous process. However, far less is known about the temporal dynamics of a bacterioplankton community responding to pollution disturbances such as toxic metals. We used coastal water microcosms perturbed with 0, 10, 100, and 1,000 μg liter⁻¹ of cadmium (Cd) for 2 weeks to investigate temporal variability, Cd-induced patterns, and their interaction in the coastal bacterioplankton community and to reveal whether the bacterial community structure would reflect the Cd gradient in a temporally varying system. Our results showed that the bacterioplankton community structure shifted along the Cd gradient consistently after a 4-day incubation, although it exhibited some resistance to Cd at low concentration (10 μg liter⁻¹). A process akin to an arms race between temporal variability and Cd exposure was observed, and the temporal variability overwhelmed Cd-induced patterns in the bacterial community. The temporal succession of the bacterial community was correlated with pH, dissolved oxygen, NO₃⁻-N, NO₂⁻-N, PO₄³⁻-P, dissolved organic carbon, and chlorophyll a, and each of these parameters contributed more to community variance than Cd did. However, elevated Cd levels did decrease the temporal turnover rate of community. Furthermore, key taxa, affiliated to the families Flavobacteriaceae, Rhodobacteraceae, Erythrobacteraceae, Piscirickettsiaceae, and Alteromonadaceae, showed a high frequency of being associated with Cd levels during 2 weeks. This study provides direct evidence that specific Cd-induced patterns in bacterioplankton communities exist in highly varying manipulated coastal systems. Future investigations on an ecosystem scale across longer temporal scales are needed to validate the observed pattern.     

Effects of Light on the Microcystin Content of Microcystis Strain PCC 7806

Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 μmol of photons m⁻² s⁻¹ on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell⁻¹ and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 μmol of photons m⁻² s⁻¹, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 μmol of photons m⁻² s⁻¹ than at 10 μmol of photons m⁻² s⁻¹ and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.     

Determination of Abundance and Biovolume of Bacteria in Sediments by Dual Staining with 4′,6-Diamidino-2-Phenylindole and Acridine Orange: Relationship to Dispersion Treatment and Sediment Characteristics

We measured the abundance and biovolume of bacteria in intertidal sediments from Tokyo Bay, Japan, by using a dual-staining technique (4′,6-diamidino-2-phenylindole and acridine orange) and several dispersion techniques (ultrasonic cleaner, ultrasonic sonicator, and tissue homogenizer). Dual staining reduced serious background fluorescence, particularly when used for silt-, clay-, and detritus-rich sediments, and allowed us to distinguish bacteria from other objects during both counting and sizing. Within the studied samples, the number of bacterial cells ranged from 0.20 × 10⁹ to 3.54 × 10⁹ g of wet sediment⁻¹. With the cleaner and sonicator treatments, the bacterial numbers for all of the sites initially increased with dispersion time and then became constant. For the homogenizer treatments, the highest bacterial numbers were observed with the shortest (0.5- to 2-min) treatments, and the counts then declined steeply as the homogenization time increased, indicating that cell destruction occurred. The cleaner treatment had the possibility of insufficient dispersion of bacteria for fine-grain sediments. Within the studied samples, the bacterial biovolume ranged from 0.07 to 0.22 μm³. With the cleaner and sonicator treatments, the biovolume peaked during the shorter dispersion time. This pattern was caused not by cell destruction but by the incremental portion of dispersed small cells. We concluded that with the cleaner and sonicator treatments, the longer dispersion time reflected the real size spectrum and was preferable for accurate estimation of mean bacterial biovolumes.     

Analysis of long-term variation in phytoplankton biovolume in the northern basin of Lake Biwa

The long-term variation in phytoplankton biovolume in the northern basin of Lake Biwa was analyzed using periodic phytoplankton census data from January 1979 to December 2009. Population densities obtained from census data were transformed into biovolumes, and phytoplankton species were categorized into three size fractions: net phytoplankton (≥4,000 μm³ cell^?1, ≥ca. 20 μm in diameter), large nanophytoplankton (100–4,000 μm³ cell^?1, ca. 6–20 μm in diameter), and small nanophytoplankton (<100 μm³ cell^?1, <ca. 6 μm in diameter). Although the annual total biovolume gradually decreased over time, the total biovolumes in winter and spring were found to increase. Furthermore, a decrease in the biovolume of net phytoplankton and an increase in that of small nanophytoplankton were observed. Because of succession in the phytoplankton community, the average cell volume of the phytoplankton community decreased from 269 μm³ cell^?1 in the 1980s to 56 μm³ cell^?1 in the 2000s. Lake warming accompanied with the intensification of thermal stratification and the augmentation of wind speed were observed at Lake Biwa over the study period. Serial analysis correcting for autocorrelation revealed that oligotrophication in the epilimnion, induced by lake warming and limitation of light available for phytoplankton growth by wind-induced water mixing, was a potential factor in the succession of the phytoplankton community.     

4-Methylumbelliferyl-β-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-β-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity β-N-acetylglucosaminidase-like enzyme (K_m, 100 μmol liter^-1) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, β-N-acetylglucosaminidase-like enzyme (K_m, <0.5 μmol liter^-1). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r² = 0.96) and Cyclidium sp. (r² = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of β-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.     

Detection and Quantification of Snow Algae with an Airborne Imaging Spectrometer

We describe spectral reflectance measurements of snow containing the snow alga Chlamydomonas nivalis and a model to retrieve snow algal concentrations from airborne imaging spectrometer data. Because cells of C. nivalis absorb at specific wavelengths in regions indicative of carotenoids (astaxanthin esters, lutein, β-carotene) and chlorophylls a and b, the spectral signature of snow containing C. nivalis is distinct from that of snow without algae. The spectral reflectance of snow containing C. nivalis is separable from that of snow without algae due to carotenoid absorption in the wavelength range from 0.4 to 0.58 μm and chlorophyll a and b absorption in the wavelength range from 0.6 to 0.7 μm. The integral of the scaled chlorophyll a and b absorption feature (I_0.68) varies with algal concentration (C_a). Using the relationship C_a = 81019.2 I_0.68 + 845.2, we inverted Airborne Visible Infrared Imaging Spectrometer reflectance data collected in the Tioga Pass region of the Sierra Nevada in California to determine algal concentration. For the 5.5-km² region imaged, the mean algal concentration was 1,306 cells ml⁻¹, the standard deviation was 1,740 cells ml⁻¹, and the coefficient of variation was 1.33. The retrieved spatial distribution was consistent with observations made in the field. From the spatial estimates of algal concentration, we calculated a total imaged algal biomass of 16.55 kg for the 0.495-km² snow-covered area, which gave an areal biomass concentration of 0.033 g/m².     

Protein Mobility in the Cytoplasm of Escherichia coli

The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809–812, 1997). The apparent diffusion coefficient, D_a, of GFP in E. coli DH5α was found to be 7.7 ± 2.5 μm²/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with D_a of 2.5 ± 0.6 μm²/s. In addition, GFP mobility can depend strongly on at least two factors: first, D_a is reduced to 3.6 ± 0.7 μm²/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces D_a to 4.0 ± 2.0 μm²/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of D_a reported here. These measurements have implications for the understanding of intracellular biochemical networks.     

Penetration of Rhizopus oligosporus into Soybeans in Tempeh

Histological observations were made on the penetration of hyphae of Rhizopus oligosporus into soybean cotyledons in tempeh, an Indonesian soybean food. Hyphal penetrations averaged one per 1,400 μm² (±390 μm²) on the curved (outer) cotyledon surface and one per 1,010 μm² (±340 μm²) on the flat (inner) one. Hyphae infiltrated to a depth of 742 μm, or about 25% of the average width of a soybean cotyledon. This previously unreported degree of penetration offers partial explanation for the rapid physical and chemical changes in soybeans during tempeh fermentation.     

Activity of the Electrogenic Pump in Chara corallina as Inferred from Measurements of the Membrane Potential, Conductance, and Potassium Permeability

The effects of various inhibitors on the membrane potential, resistance, and K⁺ permeability of Chara corallina were measured, providing evidence that there is an electrogenic pump in the membrane. It was found that: (a) 5.0 μm carbonyl cyanide m-chlorophenyl hydrazone depolarizes the membrane potential and increases the membrane resistance. This inhibition is faster in the dark than in the light but the extent of inhibition is the same in both cases. (b) Fifty μm dicyclohexylcarbodiimide increases the resistance and the K⁺ permeability and depolarizes the membrane to a diffusion potential mainly controlled by K⁺. (c) Forty μm diethylstilbestrol and 0.1 mm 2,4-dinitrophenol increase the resistance and depolarize the potential to a value given by the Goldman diffusion equation. (d) Both 3-(3,4-dichlorophenyl)-1,1-dimethylurea and darkness (at pH 6) cause the membrane resistance to increase but neither has a large effect on the potential. 3-(3,4-dichlorophenyl)-1,1-Dimethylurea increases K⁺ permeability while darkness decreases it.