Starvation of Flavobacterium psychrophilum in broth, stream water and distilled water

Physical changes in Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS), were examined over a 19 wk period of starvation. Bacteria were maintained in either Cytophaga broth, filtered stream water, or filtered distilled water, or were maintained in broth after disinfection as a negative control for dead bacteria. Culturability and viability of the bacterium were assessed using colony-forming units (CFUs) and a commercially available live/dead kit. Antigenic profiles and general morphology of the bacterium were also examined using Western blot analysis and electron microscopy, respectively. The bacterium appeared to stop multiplying and became smaller and rounded when maintained in stream water. Its culturability declined until it was no longer possible to obtain colonies on agar plates at the end of the trial at 19 wk, and results from the live/dead kit did not correspond with the viability obtained as CFUs in culture. However, it was still possible to obtain growth of the bacterium after 36 wk with a resuscitation step in Cytophaga broth. Bacteria maintained in distilled water or treated with a disinfectant appeared non-viable using the live/dead kit and were unable to grow on agar 1 h after setting up the experiment; no morphological changes were observed in the bacteria maintained under these conditions. Bacteria maintained in broth were present as long, slim rods, some of which developed into 'ring' formations. Small differences were observed in the antigen profiles of the bacteria maintained under the different treatments, possibly due to a reduction in the size and metabolism of the bacteria. There was also a marked decline in the sensitivity of the PCR with bacteria maintained under the different treatments 14 wk from the onset of the study.     

Viability and metabolic capability are maintained by Escherichia coli, Pseudomonas aeruginosa, and Streptococcus lactis at very low adenylate energy charge.

Metabolic regulation by nucleotides has been examined in several bacteria within the context of the adenylate energy charge (EC) concept. The ECs of bacteria capable of only fermentative metabolism (Streptococcus lactis and the ATPase-less mutant Escherichia coli AN718) fell to less than 0.2 under carbon-limiting conditions, but the bacteria were able to step up the EC to greater than 0.8 upon exposure to nutrient sugars. Similarly, nongrowing E. coli 25922, whose EC had been artificially lowered to less than 0.1 by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), was able to immediately step up the EC to 0.8 to 0.9 upon the addition of glucose but was unable to respond to respiratory substrates. The EC of respiring bacteria (E. coli 25922 and Pseudomonas aeruginosa 27853) fell to 0.3 to 0.4 under certain limiting growth conditions, but the bacteria also responded immediately when challenged with succinate to give EC values greater than 0.8. These bacteria could not step up the EC with respiratory substrates in the presence of CCCP. For all bacteria, the loss of the ability to step up the EC was attributable to the loss of nutrient transport function. Mixtures of viable and HOCl-killed E. coli 25922 were able to step up the EC in proportion to the fraction of surviving cells. The data indicate that nucleotide phosphorylation levels are not regulatory in nongrowing bacteria but that the EC step-up achievable upon nutrient addition may be an accurate index of viability.     

Effect of sugars and growth media on the dehydration of Lactobacillus delbrueckii ssp. bulgaricus

AIMS: The efficiency of trehalose, sucrose and maltose to protect Lactobacillus bulgaricus during drying has been evaluated in bacteria grown at low water activity. METHODS AND RESULTS: Bacteria were grown in MRS (control), and in MRS supplemented with sucrose (MRS-sucrose) or with polyethyleneglycol (PEG) (MRS-PEG) as low water activity media. The growth in low water activity media (MRS-sucrose and MRS-PEG) prior to drying enhanced the effectiveness of trehalose as thermoprotectant during drying. The efficiency of sucrose was improved when bacteria were grown in MRS-sucrose. On the other hand, the growth in both low water activity media did not affect the efficiency of maltose. The damage produced during dehydration has been evaluated by means of growth kinetics in milk. The preservation of bacteria dehydrated with sucrose, after growing them in MRS-sucrose, appears to be as efficient as the dehydration with trehalose. CONCLUSIONS: The growth of L. bulgaricus in low water activity media enhances the protective action of trehalose and sucrose. SIGNIFICANCE AND IMPACT OF THE THE STUDY: These results may aid the dairy industry to improve the recovery of the starters at low costs after preservation processes.     

Hyperbaric oxygen toxicity and ribosome destruction in Escherichia coli K12

The viability of resting suspensions of Escherichia coli K12 Ymel exposed to air plus 300 psi (1 psi = 6.895 kPa) oxygen (hyperbaric oxygen) decreased as an apparent first-order process after an initial period of constant viability. Control suspensions exposed to air plus 300 psi nitrogen (hyperbaric nitrogen) did not lose viability over the 96 h of the experiment. It was observed that a decrease in the refractive index of the cells preceded the loss of viability in hyperbaric oxygen. This finding together with electron micrographs, which showed extensive loss of ribosomal particles in bacteria incubated in hyperbaric oxygen, led us to suspect that ribosome injury or disassociation might be important in hyperbaric oxygen toxicity. In support of this we found that cellular RNA, labeled with [5-3H]uridine, was much more rapidly and more completely degraded in hyperbaric oxygen than in hyperbaric nitrogen. Furthermore, a far greater proportion of RNA was degraded than was DNA or protein. A direct assay for ribosome particles by sucrose gradient centrifugation showed that only 34% of the 70S ribosome particles was lost during the first 24 h in hyperbaric nitrogen whereas in hyperbaric oxygen 99.6% of the 70S particles was degraded during the same period. In hyperbaric oxygen the rate of viability loss between 24 and 72 h was equal to the rate of 70S ribosome degradation during the first 24 h. If 70S ribosome disassociation in hyperbaric oxygen continues at the same rate after first 24 h, then cumulative 70S ribosome disassociation or injury may lead to and provide an explanation for irreversible bacterial cell injury and the loss of viability.     

Biochemical Mechanism for Regulation of Sucrose Accumulation in Leaves during Photosynthesis

It is not known why some species accumulate high concentrations of sucrose in leaves during photosynthesis while others do not. To determine the possible basis, we have studied 10 species, known to differ in the accumulation of sucrose, in terms of activities of sucrose hydrolyzing enzymes. In general, acid invertase activity decreased as leaves expanded; however, activities remaining in mature, fully expanded leaves ranged from low (<10 micromoles per gram fresh weight per hour) to very high (>100 micromoles per gram fresh weight per hour). In contrast, sucrose synthase activities were low and relatively similar among the species (4-10 micromoles per gram fresh weight per hour). Importantly, leaf sucrose concentration, measured at midafternoon, was negatively correlated with acid invertase activity. We propose that sucrose accumulation in vacuoles of species such as soybean and tobacco is prevented by acid invertase-mediated hydrolysis. Initial attempts were made to characterize the relatively high activity of acid invertase from mature soybean leaves. Two apparent forms of the enzyme were resolved by Mono Q chromatography. The two forms had similar affinity for substrate (apparent K_m [sucrose] = 3 millimolar) and did not interconvert upon rechromatography. It appeared that the loss of whole leaf invertase activity during expansion was largely the result of changes in one of the enzyme forms. Overall, the results provide a mechanism to explain why some species do not accumulate sucrose in their leaves. Some futile cycling between sucrose and hexose sugars is postulated to occur in these species, and thus, the energy cost of sucrose production may be higher than is generally thought.     

Some observations in freeze-drying of recombinant bioluminescent Escherichia coli for toxicity monitoring

A recombinant bioluminescent bacteria, containing a fabA::luxCDABE fusion gene, has been used to characterize freeze-drying methods, which may be conveniently used as a tool for the development of a portable biosensor. Through residual water, viability, biosensing activity and scanning electron microscopy analyses, the characteristics that four cryoprotectants, trehalose, sucrose, sorbitol, and mannitol, conferred on freeze-dried samples were elucidated, including the morphology, water content and activity of the cells. It was found that trehalose showed the best freeze-drying efficiency among the tested cryoprotectants and it might have a specific capacity limitation in protection of the cells during the freeze step. Humidity might result in damage to the cells, according to the viability, when exposed to air during storage, while the water remaining post freeze-drying showed good correlation with damage to the freeze-dried cells when under air-tight storage conditions. The results with other recombinant bioluminescent bacteria indicated that these findings might be general features of the freeze-drying processes.     

Sucrose concentratidns in the growth medium influence the membrane lipids of Streptococcus mutans

Streptococcus mutans was cultivated in media containing sucrose (10–40%, w/v) and the sucrose induced changes in chemical and physical properties of its membrane lipids were investigated. The degree of unsaturation in the fatty acids of both total lipid and glycolipid fractions decreased when the sucrose concentration was increased. An electron spin resonance spectroscopic study revealed the reduction of membrane lipid fluidity by adding sucrose to the growth medium. Liposomes prepared from membrane lipids of bacteria grown with sucrose showed less osmotic volume changes than those of bacteria grown without sucrose. These results suggest that modification of membrane lipid composition, fluidity and osmosis-resistance have an important role in the ability of Streptococcus mutans to grow in sucrose at high concentrations.     

Mild disintegration methods of microalgae–bacteria flocs from wastewater treatment

The use of microalgae in biological wastewater treatment has been widely studied. However, there is a dearth of information about estimating the microalgae and bacteria concentrations. In order to maintain a stable algal-bacterial system, it is necessary to quantify both the algal and bacterial biomasses. Typically, microalgae and bacteria from flocs in activated sludge contribute to better biomass settleability. However, flocs cause problems when it comes to estimating the individual biomass concentrations of microalgae and bacteria in a symbiotic algae-bacteria aggregate. This study aimed to find the best disintegration treatment with low influence on the viability of the microalgal cell determined by its photosynthetic activity. In the present work, biological (enzyme solution), chemical (formaldehyde), mechanical (glass bead-beating), and physical (sonication) treatments were performed on microalgae–bacteria flocs (ALBA flocs) to disintegrate the community as a pre-treatment step in order to develop a method for estimating the algal and bacterial concentration and to quantify the degree of disintegration. The effectiveness of the methods to disintegrate ALBA flocs in descending order are the following: sonication, bead-beating, formaldehyde and enzyme application. Sonication treatment (40 W, 6 min) showed the best disintegration performance of the microalgal-bacterial flocs, up to 90 % with 17 % loss of the algal photosynthetic activity. Bead-beating (3 mm diameter, 80 s) achieved 80 % of disintegration with only 6 % loss of its photosynthetic activity. These results demonstrate the possibility of mild disintegration of compact ALBA flocs without having any adverse impact on the microalgae cell. After these treatments, it becomes possible to estimate the individual biomass concentrations of algae and bacteria manually such as with a cell-counting chamber.     

Toxic effects of linear alkylbenzene sulfonate on metabolic activity, growth rate, and microcolony formation of Nitrosomonas and Nitrosospira strains

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. Two Nitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC(50)], 6 to 38 mg liter(-1)) was affected much less by LAS than the growth rate and viability (EC(50), 3 to 14 mg liter(-1)) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter(-1)); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH(4)(+) oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.     

Protection by sucrose against heat-induced lethal and sublethal injury of Lactococcus lactis: an FT-IR study

The heat inactivation of Lactococcus lactis was studied by determination of cell counts, and by FT-IR spectroscopy recording the average structure of cell proteins. Cell counts were measured after incubation milk buffer or milk buffer with 1. 5 M sucrose, and FT-IR spectra were recorded in (2)H(2)O or (2)H(2)O with 1. 5 M sucrose in the range of 6-75 degrees Celsius. Sucrose protected L. lactis against heat inactivation. The cell counts differed by up to 6-log cycles after treatment in milk buffer as compared to milk buffer with sucrose. The (1)H/(2)H exchange in proteins, and secondary structure elements were detected by the analysis of amide I', amide II and amide II' bands. A reduced (1)H/(2)H exchange as well as a lower content of disordered structural elements was observed when sucrose was present. Conformational fluctuations of native proteins as indicated by the (1)H/(2)H exchange were apparent already at sublethal temperatures. The loss of viability of L. lactis occurred in the same temperature range as the loss of the protein secondary structure. These results demonstrate that sucrose protects L. lactis against heat inactivation, and that the increased heat stability of proteins in the presence of sucrose contributed to this enhanced heat resistance.     

Isolation and characterization of a glucosamine-requiring mutant of Escherichia coli K-12 defective in glucosamine-6-phosphate synthetase

A mutant was isolated from Escherichia coli K-12 which requires glucosamine or N-acetylglucosamine for growth. Depriving the mutant of glucosamine resulted in a rapid loss of viability of the cells, followed by a decrease in the turbidity of the culture. When the mutant cells were resuspended in broth media containing 10% sucrose, the rod-shaped cells became spheroplasts. However, the presence of sucrose in the media did not prevent the cells from losing their viability. This mutant was shown to be deficient in the activity of l-glutamine:d-fructose-6-phosphate aminotransferase (EC 2.6.1.16). The activity of the deaminating enzyme, 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (EC 5.3.1.10), appeared to be normal in this mutant. The position of the mutation has been determined to be at the 74th min of the Taylor and Trotter map, as shown by cotransduction with phoS (90%) and ilv (25%) by using bacteriophage P1.     

Viability of plant hairy roots is sustained without propagation in low sugar medium kept at ambient temperature

The effect of sucrose concentration in the medium on the growth and resumption ability to form lateral roots was investigated using the hairy roots of pak-bung and tobacco. It was found that the growth evaluated by root tip elongation of pak-bung and tobacco hairy roots was suppressed in the medium having an initial sucrose concentration of <2.5kg/m(3), and that the resumption abilities of both the hairy roots could be preserved when the hairy roots were kept at an initial sucrose concentration of 2.5kg/m(3) under ambient temperature conditions. The values of maintenance energy for pak-bung and tobacco hairy roots were determined to be 0.11 and 0.12 per day, respectively, from the total sugar consumption rates. Under the oligotrophic condition of the sucrose concentration of 2.5kg/m(3), the hairy roots were considered to exist as resting cells with maintenance metabolism, and the minimum demand for the energy source to ensure survival of the cells was met because the cells hardly multiplied and sugar consumption was not significant. In addition, long-term storage of pak-bung hairy roots in the liquid medium with 2.5kg/m(3) sucrose was performed at 25 degrees C. It was demonstrated that the hairy roots could maintain their resumption abilities without a serious loss of viability over 600 days and that the number of budding lateral roots per unit length of the main roots remained a value of 72 roots/m after the 600-day storage.     

Assessing the viability of three Lactobacillus bacterial species protected in the cryoprotectants containing whey and maltodextrin during freeze-drying process

Freeze-drying of bacteria associates with different stresses such as osmotic pressure, temperature and oxidation, and decreases bacterial viability, which seem to reduce by applying cryoprotectants. The present study evaluated the effect of four cryoprotectants on decreasing the stress caused by freeze-drying process among three Lactobacillus species. Additionally, it highlighted the use of whey and maltodextrin as a substitute for peptone and sucrose in cryoprotectants respectively. The viability of lactobacilli was measured after freeze-drying, 1 month of storage at 25 and 4°C. Based on the results, the viability rate of bacteria in protectants during freeze-drying stage was dependent on their strains. The best viability of Lacticaseibacillus rhamnosus GG and Ligilactobacillus salivarius 20687 was, respectively, observed in the protectants containing sucrose and whey, while Lactiplantibacillus plantarum NRRL B-14768 viability was equal in all protectants. The number of live bacteria reduced significantly by storing bacteria for 1 month at 25°C compared to the 4°C storage. During the storage period, the viability of L. salivarius improved by adding sucrose in protectant. Due to the positive effect of whey and sucrose in the drying and storage stage, on bacterial viability, the protectant consisting of whey and sucrose is suggested for all of the species under study.     

Effect of carbon source and sucrose concentration on growth and hexose accumulation of grape berries cultured in vitro

Using in vitro culture of isolated small berries of Vitis vinifera L. cv. Sultana, it was possible to study the effect of different carbon sources and sucrose concentration on fruit growth, hexose accumulation and soluble invertase activity during the first stage of berry development by eliminating the source tissue. Berries cultured in vitro lack stage III of berry development which is characterised by massive accumulation of water and sugars, and thereby berries reached only 30% of the weight of those grown in the plant. Sucrose and glucose were both good carbon sources for berry growth, while fructose was not as good. Berry growth, hexose accumulation and invertase activity increased as sucrose concentration increased up to 15% in the medium. Furthermore, the onset of hexose accumulation in cultured berries depended on the concentration of sucrose in the medium, starting earlier at higher concentrations. This revised version was published online in June 2006 with corrections to the Cover Date.     

Preservation of resuspended red cell concentrate. Red cell volume and hemolysis

Sucrose in a concentration of 30 to 50 mmol/l preservation solution (10-20 mmol/l red cell concentrate (RCC) and 3-5 mmol per unit RCC) and an ionic strength of about 0.16 avoid changes of red cell volume during 6 weeks of storage. Increasing sucrose concentrations up to 80 mmol/l RCC decrease the hemolysis. But a sucrose concentration of only 10 mmol/l RCC causes an acceptable low hemolysis rate of 0.25% after 35 days of storage in PCV FENWAL plastic bags. Sucrose can be replaced by mannitol or sorbitol at the same final concentration. Changes in red cell metabolism and viability will not be expected.     

Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides.

Leuconostoc mesenteroides NRRL B-512(F) was grown in continuous culture under conditions of energy-limited growth. The extracellular enzyme dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5), was not detected in glucose- or maltose-limited cultures. Under conditions of sucrose-limited growth, the enzyme activity of the cell-free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved. The appearance of fructose in the effluent of the sucrose-limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis. The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production. Dextransucrase was isolated from the cell-free culture supernatant by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme preparation exhibited both dextran biosynthetic activity and an invertase-like activity. The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10 degrees C. The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.     

Deoxyribonucleic Acid Breaks in Heated Salmonella typhimurium LT-2 After Exposure to Nutritionally Complex Media

Minimal medium recovery of heat-treated Salmonella typhimurium LT-2 has been expressed as the reduced viability on trypticase soy agar supplemented with 0.5% yeast extract (TSY) relative to a glucose-salts (M-9) agar. Incubation of S. typhimurium LT-2 in water at 50 C for 15 min did not change the sedimentation patterns of deoxyribonucleic acid (DNA) in alkaline sucrose gradients. The same pattern was obtained if the heated cells were further incubated for 15 min at 37 C in M-9 broth. However, a marked increase in DNA single-strand breakage accompanied by a loss of viability was observed after a similar incubation of heated bacteria in TSY broth. If heated bacteria were incubated in M-9 broth before TSY broth, there was a decrease in the single-strand breakage occurring in the TSY broth. This decrease is believed to be the result of repair of heat-induced damage. We conclude that minimal medium recovery after heat treatment is due to DNA damage caused by sequential exposure to heat and TSY medium, such damage not occurring after sequential exposure to heat and M-9 medium.     

Effect of osmolality on renal medullary Na-K-ATPase activity in the postobstructive kidney.

The aim of this study was to determine the effect of changes in osmolality on the reduced renal medullary Na-K-ATPase (EC 3.6.1.3) activity of the postobstructive kidney. The effect of osmolality on renal medullary Na-K-ATPase activity was studied by incubating tissue slices from sham-operated and bilaterally obstructed rats in media with osmolality varied before enzyme isolation using sodium chloride, choline chloride, or sucrose. Both sham-operated and bilaterally obstructed rat renal medullary enzyme showed a similar increase in activity with increased osmolality due to sodium chloride. Medullary Na-K-ATPase from the postobstructive kidney also showed increased activity with osmotic changes induced by choline chloride or sucrose. It is proposed that the decrease of Na-K-ATPase activity observed after bilateral ureteral obstruction is due, at least in part, to the loss of the solute concentration gradient in the kidney.     

Invertase,sucrose synthase and sucrose phosphate synthase in lyophilized spruce needles; microplate reader assays

Summary Data on changes of apparent activities of enzymes involved in sucrose metabolism of developing spruce needles are presented. Extractable activities of sucrose phosphate synthase (SPS, sucrose synthesis), and sucrose synthase (SS) and acid invertase (both sucrolysis) were determined in small volumes using a novel microplate reader system which combined high rates of activity with good reproducibility and high sample throughput. During a developmental period of up to 18 months after bud break characteristic changes in SPS and SS occurred. During the first 4 months of needle development SS declined while SPS increased which is indicative of a transition from net import to net export of photoassimilates (sink/source transition). After needle maturation both enzymes exhibited parallel annual changes with increasing rates towards autumn, which was mirrored by the pool sizes of sucrose (possibly due to the acquisition of frost hardiness). Acid invertase activity was comparable to that of SS but showed only marginal seasonal changes. Approximately 70% of its total activity was found to be soluble.     

Hydrophobicity of bacteria Zymomonas mobilis under varied environmental conditions

Changes in the cell surface hydrophobicity (CSH) of bacteria Zymomonas mobilis 113S were examined in response to varied environmental conditions (temperature and phase of growth, concentration or type of carbon source, the presence of amphiphilic compounds). The values of CSH were elevated with a decreased growth rate over the time of cultivation up to 20–22% at the stationary phase. CSH values increased proportionally with the growth of cultivation temperature and concentration of carbon source (glucose or sucrose) or amphiphilic compound (aliphatic alcohols, Tween80) in the medium. Replacement of sucrose by glucose and the presence of Tween20 in the growth medium resulted in reduced values of CSH. An inverse relationship was detected between the number of attached cells to the hydrophilic glass surfaces and the CSH values of Z. mobilis whereas direct linear relationship was observed for hydrophobic surfaces. Permeation rates of the fluorescent probe (NPN) into the cells were directly proportional to the concentration of extracellular protein in the medium and to the values of CSH indicating the impaired barrier function for more hydrophobic cells. The multiple correlation between the CSH values and absorption indices of FT-IR spectra at the fingerprint region (866–1088 cm⁻¹) suggests the possible contribution of carbohydrates and/or lipopolysaccharides in observed changes of Z. mobilis hydrophobicity.