Effect of starvation and chloramphenicol on acceleration of bacterial dihexyl sulfosuccinate biotransformation

Starvation for carbon and energy sources accelerated the biotransformation of the anion-active surfactant dihexyl sulfosuccinate (DHS) byComamonas terrigena cells. Cloramphenicol (Cm) added at different time intervals to non-starved cells inhibited the DHS transformation. The largest difference between cells treated and non-treated by Cm was observed for a 16-h-starvation period. Protein synthesisde novo during starvation enhanced the DHS biotransformation efficiency. A partial transformation of DHS in the presence of Cm indicated the constitutive character of enzymes involved in primary DHS biodegradation.     

Dihexyl sulfosuccinate biodegradation by mixed cultures

The primary biodegradation of dihexyl sulfosuccinate has been demonstrated using two single cultures and three mixed bacterial cultures ofComamonas terrigena. Primary biodegradation was found in all variants. The highest biotransformation rate and efficiency was observed in one of the single strains. No synergistic effect on degradation was found in mixed cultures.     

Enrichment, isolation and characterization of dialkyl sulfosuccinate degrading bacteriaComamonas terrigena N3H andComamonas terrigena N1C

Microorganisms derived from soil, sludge, sewage or river water contaminated by detergents or mineral oils were enriched and tested for the capability of primary biodegradation of dialkyl sulfosuccinates (DASS). Adapted mixed cultures, containing predominantly Gram-negative bacteria, degraded di-n-hexyl sulfosuccinate (DHSS) more readily than di-n-octyl sulfosuccinate (DOSS). Bacterial isolates obtained from enriched mixed cultures that show the highest rates of primary biodegradation and assimilation of DASS belong to the genusComamonas. Under nongrowth conditions, the DHSS primary biodegradation by these strains follows the 1st order reaction kinetics. The rate constants of primary degradation of DHSS by resting cells ofC. terrigena N3H were found to be 30 times higher than when catalyzed byC. terrigena N1C. The capacity for accelerated removal of surface-active properties of DHSS byC. terrigena N3H was retained within the range of pH 4–10 and at temperatures up to 55°C, while DHSS degradation byC. terrigena N1C was completely inhibited above 48°C. Temperature and pH limits for DHSS primary degradation by both strains were to a great extent wider than those for the viability of the cultures. Biodegradation capacity and metabolic stability ofC. terrigena N3H make this strain interesting from both scientific and technological point of view.     

Influence of clay particles (Illite) on substrate utilization by sulfate-reducing bacteria

The presence of calcium-or iron-saturated illite had a positive effect on the conversion of ethanol and acetate by non-starved cultures of Desulfobacter postgatei D.A41, but had no effect on non-starved cultures of Desulfobulbus propionicus Lindhorst and Sesulfovibrio baculatus H.L21. Starvation of these cultures at room temperature induced adhesion of cells of D. baculatus H.L21 to the surface of the clay particles. No adhesion of cells of D. propionicus Lindhorst and D. postgatei D.A41 was ever observed. However, for the three strains studied, the presence of clay particles had a positive effect on conservation of the oxidative capacity of the cultures during starvation.     

Effect of starvation upon baculovirus replication in larval Bombyxmori and Heliothisvirescens

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24 h immediately following inoculation, the median lethal time to death (LT₅₀) was delayed by 9.5-19.2 h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24 h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT₅₀ was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24 h immediately following inoculation were 10-fold lower (p < 0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24 h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.     

The effect of pre-acquisition starvation on aphid transmission of potyviruses during observed and electrically recorded stylet penetrations

The effect of pre-acquisition starvation on stylet penetration behaviour by the aphidMyzus persicae (Sulz.) and the consequent non-persistent transmission of the potyviruses beet mosaic virus (BMV) and potato virus Y (PVY) were investigated. Visual observations indicated that starved aphids initiated penetrations earlier and penetrated for shorter periods than non-starved insects. Tethering with a fine gold wire did not affect these observations with either starved or non-starved aphids, but starvation caused increased PVY and BMV acquisition efficiency, regardless of tethering. Tethered aphids were then made part of an electrical circuit and their stylet activities investigated in detail. Electrically recorded aphids also acquired and inoculated both potyviruses more efficiently when starved, and these acquisitions and inoculations were associated with stylet punctures of plant cell membranes. However, starvation did not affect the occurrence of electrically recorded membrane puncture, suggesting that non-behavioural factors may contribute to the enhancement of virus transmission by pre-acquisition starvation.     

Potential application of catalase-peroxidase from Comamonas terrigena N3H in the biodegradation of phenolic compounds

Comamonas terrigena N3H is a gram-negative rod-shaped bacterium that was isolated from contaminated soil in Slovakia. This bacterium showed remarkable biodegradation properties. We investigated the expression and functioning of two catalase isozymes in this bacterium. The typical catalase could be induced by cadmium ions, whereas the catalase-peroxidase enzyme was constitutively expressed. Since C. terrigena lacks the key enzyme for complete degradation of phenols (phenolhydroxylase), we analysed the possible removal of phenol by the two catalases of this bacterium. Addition of phenol to the culture medium led to increased expression of the catalase-peroxidase. Applying oxidative stress prior to phenol administration markedly induced the expression of the typical catalase, irrespective of the nature of the added agent. Thus, the rate of phenol degradation is rather reduced under these conditions, while growth of the cells is not impaired. We concluded that phenol peroxidation in C. terrigena can be largely attributed to the action of a catalase-peroxidase. The potential application of this enzyme in the removal of phenol from the environment is discussed.     

TNT transformation products are affected by the growth conditions of Raoultella terrigena

High concentrations of 2,4,6-trinitrotoluene (TNT) and related nitroaromatic compounds are commonly found in soil and groundwater at former explosive plants. The bacterium, Raoultella terrigena strain HB, isolated from a contaminated site, converts TNT into the corresponding amino products. Radio-HPLC analysis with [¹⁴C]TNT identified aminodinitrotoluene, diaminonitrotoluene and azoxy-dimers as the main metabolites. Transformation rate and the type of metabolites that predominated in the culture medium and within the cells were significantly influenced by the culture conditions. The NAD(P)H-dependent enzymatic reduction of nitro-substituted compounds by cell-free extracts of R. terrigena was evaluated in vitro.     

Sulphate Uptake in Two Mutants of Chlorella vulgaris with High and Low Sulphur Amino Acid Content

Two mutants of Chlorella vulgaris characterized by higher and lower content of sulphur amino acids compared with the wild strain were assayed for the efficiency of the sulphate uptake mechanism. In both mutants uptake exhibited positive cooperation kinetics and was strongly stimulated by sulphate starvation. Stimulation was depressed by cysteine and to a higher extent by methionine. Mutations affected the uptake efficiency concordantly with the level of sulphur amino acids. Addition to the starved strains of sulphate or chromate reduced the induced transport to the lower values of the non-starved strains. Addition of cycloheximide during the induction period prevented a further enhancement of transport without depressing the attained rate in the low sulphur mutant; it was followed by a rapid fall to the non-induced rate in the high sulphur mutant.     

Synthesis of unique proteins at the onset of carbon starvation inEscherichia coli

Summary Escherichia coli bulk protein synthesis continued during the first 3–4 h of carbon starvation at 50–75% that of non-starved (growing) cells. Two-dimensional gel electrophoresis analysis of in vivo pulse-labelled proteins resolved at least 30 polypeptides with new or increased synthesis, relative to total protein synthesis, during this time. Among these polypeptides were several that were also synthesized by ethanol-treatedE. coli (heat-shock proteins). In addition, a number of unique polypeptides were synthesized by carbon-starved cells. These starvation proteins may be involved in survival of the starving bacteria.     

Performance of immobilized cells for dihexyl sulfosuccinate biotransformation

Possibilities of using immobilized bacterial cells for waste water treatment in a continuous process was determined. Cells ofComamonas terrigena strain N3H immobilized in calcium alginate beads were successful by used in packed bead-type reactor for continuous biotransformation of the anion-active surfactant dihexyl sulfosuccinate. Absence of calcium ions from the treated medium led to the disruption of alginate beads within 8 d of usage. When the medium was supplemented with Ca²⁺ ions the beads were stable for at least one month in the continuous process. During the whole time period the transformation effectivity was in the range of 80–100% even at the highest, flow rate of 14 mL/min. Presented at the 4th Mini-Symposium on Biosorption and Microbial Degradation, Prague, Czech Republic, November 26–29, 1996.     

Affinity-Purification of Concanavalin A-Binding Ciliary Glycoconjugates of Starved and Feeding Tetrahymena thermophila

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.     

Comparison of Cytoplasmic pH and Cl- Influx in Cells of Chora corallina Following 'C1- Starvation'

Cytoplasmic pH and C1– influx in cells of Chara corallinahave been measured following pretreatment in C1– freesolutions (‘C1– starvation’) or in controlsolutions containing C1–. Influx of C1– was greatlyincreased following starvation for 3—24 h, but significantchanges in cytoplasmic pH were only detected after 23—24h. It is thus unlikely that increased cytoplasmic pH controlsCl-influx in the early stages of Cl—starvation. Additionof NH⁺⁴ also increased Cl- influx, both in non-starved cellsand (sometimes) in Cl-starved cells, and this effect was accompaniedby an increase in cytoplasmic pH. The possible significanceof the cytoplasmic pH and CI- concentration as factors controllingCI- influx in Chara are assessed.     

Studies in lipogenesis in vivo: Fatty acid and cholesterol synthesis during starvation and re-feeding

1. Lipogenesis in vivo has been studied in mice given a 250mg. meal of [U-¹⁴C]glucose (2·5μc) or given an intraperitoneal injection of 25μg. of [U-¹⁴C]glucose (2·0μc). 2. The ability to convert a [U-¹⁴C]glucose meal into fatty acid was not significantly depressed by 6–7hr. of starvation. In contrast, incorporation of ¹⁴C into fatty acid in the liver after the intraperitoneal dose of [¹⁴C]glucose was depressed by 80% and by more than 90% by 1 and 2hr. of starvation respectively. Carcass fatty acid synthesis from the [U-¹⁴C]glucose meal was not depressed by 12hr. of starvation, whereas from the tracer dose of [U-¹⁴C]glucose the depression in incorporation was 80% after 6hr. of starvation. 3. Re-feeding for 3 days, after 3 days' starvation, raised fatty acid synthesis and cholesterol synthesis in the liver fivefold and tenfold respectively above the levels in non-starved control mice. These increases were associated with an increased amount of both fatty acid and cholesterol in the liver. 4. After 18hr. of starvation incorporation of a [U-¹⁴C]glucose meal into carcass and liver glycogen were both increased threefold.     

Growth rate influences reductive biodegradation of the organophosphorus pesticide demeton by Corynebacterium glutamicum

The organophosphorous pesticide, demeton-S-methyl was transformed byCorynebacterium glutamicum in co-metabolism with more readilydegradable substrates. Glucose, acetate and fructose were tested as growth substrates, and the highest demeton-S-methyl biotransformation average rate (0.78 mg l^-1 h^-1) and maximum instantaneous rate (1.4 mg l^-1 h^-1) were achieved on fructose. This higher efficiency seems to be linked to the atypical behavior of C. glutamicum grown on fructose, characterized by a prolonged period of accelerating growth instead of a constant growth rate observed on glucose or acetate. More precisely, for growth rates in the 0.1–0.4 h^-1 range, a direct coupling between the specific demeton-S-methyl consumption rate and the growth rate was demonstrated on fructose during batch –, steady state continuous – or continuous cultures with a controlled transient growth rate (accelerostat technology). The demeton-S-methyl biotransformation was more favoured during an acceleration phase of the growth rate.     

IMPACT OF IRON LIMITATION ON THE PHOTOSYNTHETIC APPARATUS OF THE DIATOM CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)

Iron starvation induced marked increases in flavodoxin abundance and decreases in light-saturated and light-limited photosynthesis rates in the diatom Chaetoceros muelleri. Consistent with the substitution of flavodoxin for ferredoxin as an early response to iron starvation, increases of flavodoxin abundance were observed before declines of cell division rate or chl a specific photosynthesis rates. Changes in the abundance of flavodoxin after the addition of iron to iron-starved cells indicated that flavodoxin was not actively degraded under iron-replete conditions. Greater declines in light-saturated oxygen evolution rates than dark oxygen consumption rates indicated that the mitochondrial electron transfer chain was not affected as greatly by iron starvation as the photosynthetic electron transfer chain. The carbon:nitrogen ratio was unaffected by iron starvation, suggesting that photosynthetic electron transfer was a primary target of iron starvation and that reductions in nitrate assimilation were due to energy limitation (the C:N ratio would be expected to rise under nitrogen-limited but energy-replete conditions). Parallel changes were observed in the maximum light-saturated photosynthesis rate and the light-limited initial slope of the photosynthesis-light curve during iron starvation and recovery. The lowest photosynthesis rates were observed in iron-starved cells and the highest values in iron-replete cells. The light saturation parameter, I_k, was not affected by iron starvation, nor was the chl-to-C ratio markedly reduced. These observations were consistent with iron starvation having a similar or greater effect on photochemical charge separation in PSII than on downstream electron transfer steps. Declines of the ratio of variable to maximum fluorescence in iron-starved cells were consistent with PSII being a primary target of iron starvation. The functional cross-section of PSII was affected only marginally (<20%) by iron starvation, with the largest values observed in iron-starved cells. The rate constant for electron transfer calculated from fast repetition rate fluorescence was found to covary with the light-saturated photosynthesis rate; it was lowest in the most severely starved cells.     

Oxidative modification and breakdown of ribulose-1,5-bisphosphate carboxylase/oxygenase induced in Euglena gracitis by nitrogen starvation

When photoheterotrophic Euglena gracilis Z Pringsheim was subjected to nitrogen (N)-deprivation, the abundant photosynthetic enzyme ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was rapidly and selectively degraded. The breakdown began after a 4-h lag period and continued for a further 8 h at a steady rate. After 12 h of starvation, when the amount of Rubisco was reduced to 40%, the proteolysis of this enzyme slowed down while degradation of other proteins started at a similar pace. This resulted in a decline of culture growth, chloroplast disassembly — as witnessed by chlorophyll (Chl) loss — and cell bleaching. Experiments with spectinomycin, an inhibitor of chloroplastic translation, indicated that there was an absolute increase in the rate of Rubisco degradation in the N-deprived culture as compared with control conditions, where no significant carboxylase breakdown was detected. Oxidative aggregation of Rubisco (as detected by non-reductive electrophoresis) and association of the enzyme to membranes increased with time of N-starvation. Fluorescent labeling of oxidized cysteine (Cys) residues with monobromobimane indicated a progressive oxidation of Cys throughout the first hours of N-deprivation. It is concluded that Rubisco acts as an N store in Euglena, being first oxidized, and then degraded, during N-starvation. The mobilization of Rubisco allows sustained cell growth and division, at almost the same rate as the control (non-starved) culture, during 12 h of N-deprivation. Afterwards, breakdown is extended to other photosynthetic structures and the whole chloroplast is dismantled while cell growth is greatly reduced.Abbreviations Chl chlorophyll - Cys cysteine - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate We thank Drs. Pablo Vera and Ismael Rodrigo (Univ. Politécnica, Valencia, Spain) for advice and facilities in raising and collecting the anti-Rubisco serum. This work was supported by grants PB87-0353 and PB92-0821 of DGICYT and by a fellowship of the Spanish Ministerio de Educación y Ciencia (awarded to C.G.-F.).     

The Expression and Diversity of Catalases in Isolates of Genus Comamonas in Response to the Oxidative Stress of a Polluted Environment

We have evaluated the role of monofunctional heme-containing catalase encoded by cat-1 gene from the soil bacterium Comamonas terrigena N3H in the response to various forms of oxidative stress. Our results indicate that this constitutively expressed catalase represents the major source for the defence of Comamonas terrigena cells against toxic peroxides but the cells can express also a second form of catalase that is bigger and its regulation is probably more complicated. The sequence analysis confirmed the presence of highly conserved catalase sequence motifs in two environmental strains of Comamonas terrigena but in those strains that were not exposed to oxidative stress, no such sequence motif could be detected. The results obtained underline the importance of catalase expression in the defence mechanism against oxidative stress in bacterial cells.     

Starvation effects on the ultrastructure of amoeba mitochondria

Summary Mitochondria in non-starved giant amoebae, Pelomyxa carolinensis, contain tubules lying at random in the matrix. Many mitochondria in starved amoebae have enlarged tubules aligned in a zigzag pattern. Tubules within the zigzag region are separated by very little matrix material. Some of these altered mitochondria are found in 70% of amoebae starved for only 24 hours, and in nearly all P. carolinensis starved for 8 days or longer. The percentage of such altered mitochondria increases from zero in most well-fed amoebae, to about 60% after two weeks of continuous starvation. Most P. carolinensis starved at 25° C survive less than three weeks. Microfilament bundles are observed in the matrix of some mitochondria in amoebae starved for more than two days.Work supported by the U. S. Atomic Energy Commission.The authors acknowledge the assistance of Miss Doris Jean Buer and Miss Patricia Ann Sustarsic.     

Respiration and ammonia excretion of the shrimpCrangon crangon L.: Metabolic response to prolonged starvation

Summary Oxygen consumption and ammonia excretion were measured simultaneously in 25 individual shrimps (C. crangon) every two days during a 30-day imposed starvation.The first response to starvation was a 10% decrease in O₂ consumption and a 25% decrease in ammonia excretion. Following this, O₂ consumption decreased sharply while ammonia excretion tended to rise. From the 14th day until the end of the experiment, the rates of each remained steady, the respiratory rate being about 60% below and rate of excretion about 30% above control values.From the O:N ratio it appeared that carbohydrate reserves were quickly exhausted (3–4 days) and that lipids and proteins were the main substrates oxidized to meet the energetic requirements ofC. crangon. After 2 weeks of starvation the O:N ratio remained constant near a value of 8, indicating that only proteins were being utilised; a 50% loss of body protein after 30 days suggested that structural proteins were heavily catabolized. The meatabolic response ofCrangon to starvation (although seasonal variations must be considered) appears to depend mainly on high nitrogen requirements.