Uptake of leucine by a marine Gram-negative heterotrophic bacterium during exposure to starvation conditions

The uptake of leucine by S14, an unidentified marine Gram-negative bacterium, was studied during a starvation period of 96 h. The S14 cells displayed two separate uptake systems with different affinities for leucine. The K_m values of these systems were 0.76 μM and 20 μM, respectively. The time of exposure to starvation had a marked effect on both uptake systems, not by changing the affinity for leucine, but rather by altering the velocity of uptake (V_max). A marked increase in the uptake capacity was noted for the high-affinity system, whereas the uptake velocity decreased for the low-affinity system. An osmotic shock treatment resulted in an almost complete loss of substrate binding activity. A gradual recovery of the leucine uptake subsequent to the osmotic shock was observed during a 72-h period of starvation. Separation of the osmotic shock supernatant by gel filtration revealed two proteins, 37 and 44 kDa in size, with leucine binding activity.     

Effect of Interfaces on Small, Starved Marine Bacteria

The copiotrophic marine Vibrio sp. strain DW1, shown previously in batch culture to increase in numbers at the onset of starvation and then to form viable small cells with low endogenous respiration, appears to have a survival advantage at interfaces. Vibrio sp. strain DW1 behaved differently at interfaces compared with the aqueous phase under starvation conditions: (i) small cells were observed at an air-water interface without nutrients, (ii) nutrients added to the air-water interface quickly produced larger cells at the surface, (iii) motility persisted many hours longer at the solid-water interface of a dialysis membrane in a microchamber at the onset of starvation, and (iv) regrowth and division at the solid-liquid interface occurred quickly and at nutrient concentrations too low to permit growth in the aqueous phase. It was concluded that, if small starved cells from copiotrophic bacteria can reach an interface, additional survival mechanisms become available to them: (i) interfaces constitute areas of favorable nutrient conditions, and (ii) interfaces lacking a sufficient amount of nutrient, nevertheless, trigger cells to become smaller, thus increasing their surface/volume ratio and the packing density.     

Starvation-Induced Effects on Bacterial Surface Characteristics

Changes in bacterial surface hydrophobicity, charge, and degree of irreversible binding to glass surfaces of seven marine isolates were followed during starvation. The degree of hydrophobicity was measured by hydrophobic interaction chromatography and by two-phase separation in a hexadecane-water system, whereas changes in charge were measured by electrostatic interaction chromatography. All isolates underwent the starvation-induced responses of fragmentation, which is defined as division without growth, and continuous size reduction, which results in populations with increased numbers of smaller cells. The latter process was also responsible for a significant proportion of the total drop in cell volume; this was observed by noting the biovolume (the average cell multiplied by the number of bacteria) of a population after various times of starvation. Four strains exhibited increases in both hydrophobicity and irreversible binding, initiated after different starvation times. The most hydrophilic and most hydrophobic isolates both showed a small increase in the degree of irreversible binding after only 5 h, followed by a small decrease after 22 h. Their hydrophobicity remained constant, however, throughout the entire starvation period. On the other hand, one strain, EF190, increased its hydrophobicity after 5 h of starvation, although the degree of irreversible binding remained constant. Charge effects could not be generally related to the increase in irreversible binding. Scanning electron micrographs showed a large increase in surface roughness throughout the starvation period for all strains that showed marked changes in physicochemical characteristics.     

Computerized Determination of Adipocyte Size

Objective: Fat cell size is a fundamental parameter in the study of adipose tissue metabolism, because it markedly influences the cellular rates of metabolism. Previous techniques for the sizing of adipocytes are often complicated or time‐consuming. The aim of this study was to develop a new, computerized method for rapid and accurate determination of adipocyte size in a cell suspension obtained by incubating human or rat adipose tissue biopsies with collagenase. Research Methods and Procedures: The cell suspension was placed between a siliconized glass slide and a cover slip. Using the reference method [designated as (R)], the cell diameters were determined manually using a microscope with a calibrated ocular. The new method presented here [designated as (C)] was based on computerized image analysis. Results: After two well‐defined corrective adjustments, measurements with (R) and (C) agreed very well. The small remaining differences seemed, in fact, to depend on inconsistencies in (R). Discussion: We propose that (C) constitutes a valuable tool to study fat cell size, because this method is fast and allows the assessment of a sufficient number of cells to get reliable data on size distribution. Furthermore, images of cell preparations may be stored for future reference.     

Construction and use of a new vector/transposon, pLBT::mim-Tn10:lac:kan, to identify environmentally responsive genes in a marine bacterium

Abstract The previously described pLOFKm transposon delivery plasmid (J. Bacteriol. (1990) 172, 6557–6567) was engineered such that a promoterless lacZ gene was cloned within the transposon cassette, generating the vector pLBT. Using pLBT, stable insertion mutations were generated at high frequencies in Vibrio sp. S141 and Pseudomonas sp. S91, and the interrupted genes could be monitored for their pattern of regulation. Genetic screens isolated mutants defective in a variety of activities. We describe the construction and use of pLBT as a tool for reporter gene mutant analysis in bacteria other than well-characterized laboratory strains.     

Population dynamics of microtine rodents: an experimental test of the predation hypothesis

One of the most popular hypotheses to explain the 3–5 year vole cycle in Fennoscandia is the predation hypothesis, which emphasises the role of specialist predators, especially small mustelids, in driving the microtine rodent cycle. I have tested this hypothesis in a field experiment with a focus on the interaction between the least weasel ( Mustela nivalis nivalis L.) and the field vole ( Microtus agrestis L.). The test involved a perturbation of the natural vole-weasel dynamics by increasing the numbers of predators. In natural cyclic dynamics, the numbers of weasels follow the numbers of voles with a time lag; the delay in the numerical response of the predators essentially leads to the cyclic dynamics. In this experiment, I attempted to eliminate the time lag in predators' numerical response by adding weasels into the system at a point when vole numbers are increasing but weasels are still scarce. The experiment was conducted in three experimental islands (each 5–10 km²) using adjacent comparable islands as controls. Results of the experiment did not provide strong evidence either for or against the hypothesis. Two of three experimental areas did not show a treatment effect, while one area showed an effect in both population dynamics and population structure. Unexpectedly, the previously clearly cyclic dynamics were much less regular in the study areas (and elsewhere in southern Finland) during the period of the experiment.     

Genetic assessment of spatiotemporal evolutionary relationships and stocking effects in grayling (Thymallus thymallus, Salmonidae)

Domestically reared introduced (or escaped) individuals can have detrimental genetic effects on the indigenous populations into which they are released. Consequently, numerous studies have attempted to estimate whether non-native specimens have contributed to the gene pool of wild populations. So far, the key limiting factor of such studies has been their lack of appropriate baseline genetic material. Here, microsatellite DNA analyses of historical scale samples and contemporary wild and introduced populations were used to assess spatiotemporal population structure and stocking effects among endangered Lake Saimaa (eastern Finland) grayling ( Thymallus thymallus , Salmonidae). Significant decreases in genetic differentiation were detected between wild and introduced populations since the commencement of stocking in 1986. Accordingly, up to 15% of the contemporary wild grayling were confidently identified to be of pure hatchery origin, and recent hybridization between the hatchery and indigenous individuals appeared likely. Despite these clear genetic imprints of stocking, the contemporary populations exhibited evolutionary relationships congruent with the sampling locations, and up to 73% of contemporary individuals were identified to be of pure indigenous origin. The use of historical baseline material should prove efficient for monitoring gene flow between domesticated and wild populations in other species also, e.g. salmonids, game animals and plants.     

A vegetable oil feeding history affects digestibility and intestinal fatty acid uptake in juvenile rainbow trout Oncorhynchus mykiss

Future expansion of aquaculture relies on the use of alternatives to fish oil in fish feed. This study examined to what extent the nature of the feed oil affects intestinal lipid uptake properties in rainbow trout. The fish were fed a diet containing fish (FO), rapeseed (RO) or linseed (LO) oil for 8 weeks after which absorptive properties were assessed. Differences in digestibility due to feed oil history were measured using diet FO with an indigestible marker. Intestinal integrity, paracellular permeability, in vitro transepithelial fatty acid transport (³H-18:3n − 3 and ¹⁴C-16:0) and their incorporation into intestinal epithelia were compared using Ussing chambers. Feed oil history did not affect the triacylglycerol/phosphatidylcholine ratio (TAG/PC) of the newly synthesized lipids in the segments. The lower TAG/PC ratio with 16:0 (2:1) than with 18:3 (10:1) showed the preferential incorporation of 16:0 into polar lipids. The FO-feeding history decreased permeability and increased transepithelial resistance of the intestinal segments. Transepithelial passage rates of 18:3n − 3 were higher when pre-fed LO compared to RO or FO. Similarly, pre-feeding LO increased apparent lipid and fatty acid digestibilities compared to RO or FO. These results demonstrate that the absorptive intestinal functions in fish can be altered by the feed oil history and that the effect remains after a return to a standard fish oil diet.