Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation

The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the same levels as found during growth.Nonstandard Abbreviations cGMP 3,5 guanosine monophosphate - ppGpp guanosine tetraphosphate - MS mineral salts - HC casein hydrolysate - TEA triethanolamine buffer - Pi inorganic phosphate     

Adenosine triphosphate pool levels and endogenous metabolism in Arthrobacter crystallopoietes during growth and starvation

The adenosine triphosphate (ATP) content of Arthrobactery crystallopoietes was measured during growth, starvation and recovery from starvation. During exponential growth of the cells as spheres in a glucose salts medium, the level of ATP per cell remained constant at 8.0×10^-10 g/cell. Morphogenesis to rodshaped cells and an increased growth rate following addition of casein hydrolysate was accompanied by an almost two-fold increase in the ATP level. As division of the rod-shaped cells proceeded, the level of ATP declined. After growing as rods for 12–14 h the cells underwent fragmentation to spheres during which time the ATP level again increased to the original value of 8.0×10^-10 g/cell. As the spherical cells resumed growth on the residual glucose, their ATP content declined for a short period and then remained relatively constant. During starvation of sphere or rod-shaped cells for one week, the ATP level declined by approximately 70% during the first 40–50 h and then remained constant. The endogenous metabolism rate of spherical cells declined during the first 10–20 h of starvation and then remained constant at approximately 0.02% of the cell carbon being utilized per h. Addition of glucose to spherical cells which had been starved for one week increased both the ATP content per cell and their rate of endogenous metabolism. The ATP content fluctuated and then remained at a level higher than maintained during starvation while endogenous metabolism quickly declined.Non-Standard Abbreviations ATP adenosine triphosphate - GS glucose mineral salts - HC casein hydrolysate - PVP polyvinylpyrrolidone - DMSO dimethylsulfoxide - MOPS morpholinopropane sulfonic acid - EDTA ethylene diaminetetraacetic acid     

Long-Term Starvation Survival of Rod and Spherical Cells of Arthrobacter crystallopoietes

Spherical and rod-shaped cells of Arthrobacter crystallopoietes, harvested during exponential growth, were subjected to total starvation for periods of time as long as 80 days. Viability measurements were made by plate count and slide culture procedures. Both cell forms remained 100% viable for 30 days. Thereafter, viability of rods and spheres decreased equally at a slow rate. After 60 days of starvation, more than 65% of both cell forms were viable. No significant cell lysis occurred as evidenced by microscopic examination, the small amount of 260-nm absorbing material found in the starvation buffer, and stability of radioactively labeled deoxyribonucleic acid in the cells. Endogenous respiration decreased 80-fold during the first 2 days of starvation, accompanied by a 30% decrease in dry weight of the cells. Thereafter, cellular carbon was oxidized to CO(2) at the constant level of 0.03%/hr over the remaining 78-day starvation period.     

Biochemical changes accompanying the long-term starvation of Micrococcus luteus cells in spent growth medium

Changes in the biochemical properties of Micrococcus luteus cells were studied during the transition to a dormant state after incubation in an extended stationary phase. The overall DNA content after 150 days of starvation was similar to its initial level, while the RNA content decreased by 50%. Total lipids and protein, phospholipids and membrane proteins declined rapidly within the first 1–10 days of starvation. After 180 days of starvation, cells contained 43% of the protein and 35% of the lipid initially present. Starvation for 120 days resulted in the loss of phosphatidylglycerol and, to some extent, of phosphatidylinositol, giving a membrane whose phospholipids consisted mainly of cardiolipin. The membrane fluidity declined during starvation, as judged by diphenyl hexatriene fluorescence anisotropy measurements. Oxidase activities declined to zero within the first 20–30 days of starvation, while the dehydrogenases and cytochromes were more stable. The activities of some cytoplasmic enzymes were lost very rapidly, while NADPH-linked isocitrate dehydrogenase had 30% of its initial activity after 120 days of starvation. For all parameters tested there were significant fluctuations during the first 10–20 days of starvation, which may reflect cryptic growth in the culture.Abbreviations MPN Most probable number - DPH Diphenyl hexatriene     

Ethylene- and enzyme-mediated superoxide production and cell death in carrot cells grown under carbon starvation

Carrot (Daucus carota L.) suspension cells are known to exhibit acclimation responses, such as fatty acid catabolism, during the first 5 days of carbon starvation. The present study shows that cells start to die once starvation is prolonged beyond this period. We investigated the signals involved in this transition from acclimation to cell death. Significant amounts of superoxide were produced transiently at days 4-5 (after the initiation of starvation). When diphenylene iodonium, an inhibitor of NADPH oxidase and/or other ROS (reactive oxygen species)-generating enzymes, was added to the cell suspension at the beginning of the starvation period, superoxide production and cell death, as measured by internucleosomal cleavage, were effectively inhibited, indicating that NADPH oxidase and/or other ROS-generating enzymes are involved in the production of the superoxide during starvation. Ethylene, which is known to be produced transiently at day 1 in these cells, positively regulated production of the superoxide and cell death, indicating that ethylene is an upstream regulator of NADPH oxidase or other ROS-generating enzymes. We suggest that as the carrot cells are dying under conditions of carbon starvation they put the following signal relays into operation: ethylene production M activation of NADPH oxidase (or other ROS-generating enzymes) M superoxide production M internucleosomal cleavage (cell death).     

Anaerobic baffled reactor (ABR) for treating heavy oil produced water with high concentrations of salt and poor nutrient

The start-up and operational performance (total 212 days, including the start-up of 164 days) of an anaerobic baffled reactor (ABR), which is used to treat heavy oil produced water, was studied without the temperature control. Inoculums were mixtures of acclimated sediment taken from a heavy oil produced water treatment plant and digested sludge from a sewage wastewater treatment plant. The rod-shaped and spherical granules with colors of henna and black, in which Clostridia, Methanosarcina and Methanothrx sp. were main populations, were observed in each compartment of ABR after the reactor's successful start-up (day 164). Rhodopseudomonas with the activity of lipase and halotolerant, as a kind of photosynthetic bacteria, was also observed in the first five compartments. X-ray diffraction (XRD) showed that the spherical granule sludge was compact and contained a large amount of organics, amorphous materials, and crystals of Fe(2)O(3), FeS, and CaCO(3), whereas the rod-shaped granule sludge was incompact without crystals of Fe(2)O(3), FeS, and CaCO(3). Scanning electron microscope (SEM) showed that the skeleton construction of this rod-shaped granule was filamentous bacteria and amount of extracellular polymeric substances (EPS). The ABR, after successful start up, can achieve high average chemical oxygen demand (COD) and oil removals of 65% and 88% for heavy oil produced water with poor nutrient (COD:TN:TP, 1200:15:1) and high salt concentration (1.15-1.46%), respectively. Furthermore, ABR kept stable during 2.5 times the COD level shock load (0.50 kg COD m-3 d-1) for four days.     

Lipid body formation plays a central role in cell fate determination during developmental differentiation of Myxococcus xanthus

Cell differentiation is widespread during the development of multicellular organisms, but rarely observed in prokaryotes. One example of prokaryotic differentiation is the Gram-negative bacterium Myxococcus xanthus . In response to starvation, this gliding bacterium initiates a complex developmental programme that results in the formation of spore-filled fruiting bodies. How the cells metabolically support the necessary complex cellular differentiation from rod-shaped vegetative cells into spherical spores is unknown. Here, we present evidence that intracellular lipid bodies provide the necessary metabolic fuel for the development of spores. Formed at the onset of starvation, these lipid bodies gradually disappear until they are completely used up by the time the cells have become mature spores. Moreover, it appears that lipid body formation in M. xanthus is an important initial step indicating cell fate during differentiation. Upon starvation, two subpopulations of cells occur: cells that form lipid bodies invariably develop into spores, while cells that do not form lipid bodies end up becoming peripheral rods, which are cells that lack signs of morphological differentiation and stay in a vegetative-like state. These data indicate that lipid bodies not only fuel cellular differentiation but that their formation represents the first known morphological sign indicating cell fate during differentiation.     

Intracellular Substrates for Endogenous Metabolism During Long-Term Starvation of Rod and Spherical Cells of Arthrobacter crystallopoietes

Cells of Arthrobacter crystallopoietes, harvested during growth as spheres and as rods, were starved by shaking at 30 C in phosphate buffer for 30 days, during which time they maintained 100% viability. Changes in cellular components and the activity of specific enzyme pathways were monitored. A glycogen-like polysaccharide comprised 40% of the dry weight of growing spherical cells and 10% of the dry weight of rod cells. This material was utilized at approximately the same rate, on a percentage basis, during starvation of both cell forms. The rods degraded intracellular protein at approximately twice the rate of the spheres. At the end of 30 days, the rods had degraded 40% and the spheres 20% of their initial content of protein. Ribonucleic acid (RNA) was degraded significantly more rapidly in the rods. After 30 days starvation, 85 and 32% of the initial RNA of rods and spheres, respectively, had been depleted. Magnesium ion followed this same general pattern; the rods lost 65% and the spheres 45% of their initial content during 28 days of starvation. Deoxyribonucleic acid increased by 20% during the first few hours of starvation of both cell forms and then remained constant. The ability of glucose-, succinate-, and 2-hydroxypyridine (2-HP)-grown cells to oxidize glucose remained constant during 14 days of starvation. The ability of succinate-grown cells to oxidize succinate decreased rapidly during the first few hours of starvation to a rate which remained constant for 14 days. Cells adapted to growth on 2-HP completely lost their ability to oxidize this substrate after 3 days starvation.     

A common step for changing cell shape in fruiting body and starvation-independent sporulation of Myxococcus xanthus

Myxococcus xanthus can sporulate in either of two ways: at the end of the program of fruiting body development or after exposure of growing cells to certain reagents such as concentrated glycerol. Fruiting body sporulation requires starvation, while glycerol sporulation requires rapid growth, and since the two types of spores are structurally somewhat different, it has generally been assumed that the two processes are different. However, a Tn5 Lac insertion mutation, Omega7536, has been isolated which simultaneously blocks the development of fruiting body spores as well as glycerol-induced spores. Both sporulation pathways are blocked in the mutant within the process that converts a rod-shaped cell into a spherical spore. The Omega7536 locus is expressed at the time of cell shape change appropriate to each process, early after glycerol induction and late after starvation induction. On the C-signal response pathway, it is possible to identify positions for the normal function of the Omega7536 locus and for the inducing stimulus from glycerol that are unique and consistent with the observations. Although the two sporulation pathways differ in certain respects, it is shown that they share at least one step for changing a rod-shaped cell into a spherical spore.     

Responses of Marine Bacteria Under Starvation Conditions at a Solid-Water Interface

Size changes during starvation of 17 marine bacterial isolates at a solid-water interface and in the liquid phase were examined. Twelve rod-shaped, hydrophilic bacteria decreased in size more rapidly at the solid surface than in the liquid phase, a result parallel to that observed previously for one of the strains at an air-water interface. On the other hand, three rod-shaped, hydrophobic bacteria diminished in size more rapidly in the liquid phase than at the solid-water interface. The rapid size decrease (defined here as the dwarfing phase) in either situation appeared to be an active process which occurred more rapidly when the cells were in an early stage of logarithmic growth at the onset of starvation. Dwarfing was reversibly inhibited by low temperature and low pH but was not inhibited by chloramphenicol. Three coccoidal bacteria showed little tendency to become smaller upon starvation in the liquid phase or at a surface.     

Starvation-Survival Processes of a Marine Vibrio

Levels of DNA, RNA, protein, ATP, glutathione, and radioactivity associated with [³⁵S]methionine-labeled cellular protein were estimated at various times during the starvation-survival process of a marine psychrophilic heterotrophic Vibrio sp., Ant-300. Values for the macromolecules were analyzed in terms of total, viable, and respiring cells. Electron micrographs (thin sections) were made on log-phase and 5.5-week-starved cells. On a per-cell basis, the levels of protein and DNA rapidly decreased until a constant level was attained. A second method in which radioactive sulfur was used for monitoring protein demonstrated that the cellular protein level decreased for approximately 2.5 weeks and then remained constant. An initial decrease in the RNA level with starvation was noted, but with time the RNA (orcinol-positive material) level increased to 2.5 times the minimum level. After 6 weeks of starvation, 45 to 60% of the cells remained capable of respiration, as determined by iodonitrotetrazolium violet-formazan granule production. Potential respiration and endogenous respiration levels fell, with an intervening 1-week peak, until at 2 weeks no endogenous respiration could be measured; respiratory potential remained high. The cell glutathione level fell during starvation, but when the cells were starved in the presence of the appropriate amino acids, glutathione was resynthesized to its original level, beginning after 1 week of starvation. The cells used much of their stored products and became ultramicrocells during the 6-week starvation-survival process. Ant-300 underwent many physiological changes in the first week of starvation that relate to the utilization or production of ATP. After that period, a stable pattern for long-term starvation was demonstrated.     

Synthesis of phosphatidylserine in carrot cells cultured under carbon-source starvation

When carrot suspension cells were cultured on medium containing no carbon source (starvation), the levels of phosphatidylserine (PS) increased transiently 3-4 d after the initiation of starvation while levels of most other phospholipid (PL) species decreased. We previously reported that fatty acids of these PLs served as an alternative carbon source during starvation. The present study showed that cells possess two different biosynthetic pathways involving phosphatidylcholine (PC)/phosphatidylethanolamine (PE) exchange enzymes and PS synthase to synthesize PS. These activities peaked similarly 4 d after the initiation of starvation and coincided with the peak of PS level. The synthesis of serine was also significantly activated during starvation. The activity of phosphoserine aminotransferase (PSAT) which is involved in serine synthesis increased with a time course similar to that of the increase in the PS level. These observations suggest that the increase in PS level plays an important role in membranes which are degraded during starvation.     

The Proteomic Response of Bacillus pumilus Cells to Glucose Starvation

Since starvation for carbon sources is a common condition for bacteria in nature and it can also occur in industrial fermentation processes due to mixing zones, knowledge about the response of cells to carbon starvation is beneficial. The preferred carbon source for bacilli is glucose. The response of Bacillus pumilus cells to glucose starvation using metabolic labeling and quantitative proteomics was analyzed. Glucose starvation led to an extensive reprogramming of the protein expression pattern in B. pumilus. The amounts of proteins of the central carbon metabolic pathways (glycolysis and TCC) remained stable in starving cells. Proteins for gluconeogenesis were found in higher amounts during starvation. Furthermore, many proteins involved in acquisition and usage of alternative carbon sources were present in elevated amounts in starving cells. Enzymes for fatty acid degradation and proteases and peptidases were also found in higher abundance when cells entered stationary phase. Among the proteins found in lower amounts were many enzymes involved in amino acid and nucleotide synthesis and several NRPS and PKS proteins.     

Variations in rRNA content of marine Vibrio spp. during starvation-survival and recovery.

The degree and temporal context of variations in ribosome content during nutrient starvation of two copiotrophic marine bacteria, Vibrio alginolyticus and Vibrio furnissii, have been examined. The organisms were starved either by nutritional shift-down or by consumption of limiting nutrients resulting from growth into stationary phase. Measurements of the amount of hybridization to 16S rRNA-specific probes revealed that the cells retained between 10 and 26% of their original rRNA content after 15 days of starvation. In V. alginolyticus, losses in stationary-phase cells occurred rapidly (1 to 2 days), whereas cells shifted into starvation remained larger and retained considerably more rRNA. The ability of V. alginolyticus to recover from starvation was assessed after cells were maintained for 2, 8, and 15 days in nutrient-depleted medium. The pattern of recovery at the level of rRNA accumulation depended upon the duration of nutrient deprivation and the manner in which it was imposed. Stationary-phase cells starved for 2 days had only slight relative increases in rRNA levels after excess nutrients were added. As the duration of starvation lengthened to 8 and 15 days, increasingly greater amounts of rRNA (30 and 70 times preenrichment values, respectively) were transcribed after nutrient enrichment. Shift-down cells recovered from 2 and 8 days of starvation without extensive rRNA production. After 15 days, nutrient enrichment caused 16S rRNA levels to increase 30-fold. The results indicate that the mechanisms controlling starvation-survival in these marine bacterial species are linked to the physiological state at the onset of starvation and that the subsequent pattern of recovery will depend upon how starvation was initiated.     

Variations in rRNA content of marine Vibrio spp. during starvation-survival and recovery.

The degree and temporal context of variations in ribosome content during nutrient starvation of two copiotrophic marine bacteria, Vibrio alginolyticus and Vibrio furnissii, have been examined. The organisms were starved either by nutritional shift-down or by consumption of limiting nutrients resulting from growth into stationary phase. Measurements of the amount of hybridization to 16S rRNA-specific probes revealed that the cells retained between 10 and 26% of their original rRNA content after 15 days of starvation. In V. alginolyticus, losses in stationary-phase cells occurred rapidly (1 to 2 days), whereas cells shifted into starvation remained larger and retained considerably more rRNA. The ability of V. alginolyticus to recover from starvation was assessed after cells were maintained for 2, 8, and 15 days in nutrient-depleted medium. The pattern of recovery at the level of rRNA accumulation depended upon the duration of nutrient deprivation and the manner in which it was imposed. Stationary-phase cells starved for 2 days had only slight relative increases in rRNA levels after excess nutrients were added. As the duration of starvation lengthened to 8 and 15 days, increasingly greater amounts of rRNA (30 and 70 times preenrichment values, respectively) were transcribed after nutrient enrichment. Shift-down cells recovered from 2 and 8 days of starvation without extensive rRNA production. After 15 days, nutrient enrichment caused 16S rRNA levels to increase 30-fold. The results indicate that the mechanisms controlling starvation-survival in these marine bacterial species are linked to the physiological state at the onset of starvation and that the subsequent pattern of recovery will depend upon how starvation was initiated.     

Survival of Vibrio parahaemolyticus at low temperatures under starvation conditions and subsequent resuscitation of viable, nonculturable cells.

Morphological changes of Vibrio parahaemolyticus from rods to spheres took place after a culture was subjected to starvation at a wide range of temperatures. Scanning electron micrographs revealed that starved spherical cells gradually developed a rippled cell surface with blebs and an extracellular filamentous substance adhesive to the cell surface. Cells starved at a low temperature for certain intervals were counted by various bacterial enumeration methods, including plate count, direct viable count, and total cell count for both Kanagawa-positive and -negative strains. The results indicated that this species could reach the nonculturable stage in 50 to approximately 80 days during starvation at 3.5 degrees C. Kanagawa-negative strain 38C6 lost culturability more slowly than Kanagawa-positive strain 38C1 at low temperature. As detected by thiosulfate-citrate-bile salts-sucrose plate count, a high percentage of the surviving cells at 3.5 degrees C in starvation medium were possibly injured by the low temperature rather than by starvation. Both addition of nalidixic acid to the starved cultures and the most-probable-number method demonstrated that the cells recovered after a temperature upshift probably represented the regrowth of a few surviving cells. These surviving cells were capable of growth and multiplication with limited nutrients at an extraordinary rate when the temperature was upshifted.     

Starvation and nutrient resuscitation of Klebsiella pneumoniae isolated from oil well waters

Klebsiella pneumoniae isolated from oil well waters reduced in size in response to nutrient starvation. The cells remained viable during starvation and later were able to grow rapidly when stimulated by nutrients. The heterotrophic potential, culture absorbance and extracellular polysaccharide production decreased during cell starvation whereas an initial increase in colony-forming units was observed on agar plates. Transmission electron microscopy (TEM) after 24 d revealed that the cells had changed to small rods or cocci between 0.5 by 0.25 micron and 0.87 by 0.55 micron. When transferred to half-strength brain heart infusion medium, TEM showed cell division and rod-shaped cells after 45 min and full resuscitation within 4 h. Cell response was much slower in sodium citrate medium and resuscitation took 8 h.     

Isolation and phenotypic characterization of Myxococcus xanthus mutants which are defective in sensing negative stimuli.

Myxococcus xanthus is a gram-negative gliding bacterium that exhibits a complex life cycle. Exposure of M. xanthus to chemicals like dimethyl sulfoxide (DMSO) at nondeleterious concentrations or the depletion of nutrients caused several negative responses by the cells. DMSO (> 0.1 M) or nutrient depletion triggered a repellent response: cell swarming was inhibited and FrzCD (a methyl-accepting chemotaxis protein) was demethylated; higher concentrations of DMSO (> 0.3 M) or prolonged starvation induced an additional response which involved cellular morphogenesis: DMSO caused cells to convert from rod-shaped vegetative cells to spherical, environmentally resistant "DMSO spores," and starvation induced myxospore formation in the fruiting bodies. In order to investigate the nature of these responses, we isolated a number of mutants defective in negative chemotaxis and/or sporulation. Characterization of these mutants indicated that negative chemotaxis plays an important role in colony swarming and in developmental aggregation. In addition, the results revealed some of the major interrelationships between the signal transduction pathways which respond to negative stimuli: (i) DMSO exposure and starvation were initially sensed by different systems, the neg system for DMSO and the stv system for starvation; (ii) the repellent response signals triggered by DMSO or starvation were then relayed by the frz signal transduction system; mutants defective in these responses showed altered FrzCD methylation patterns; and (iii) the morphogenesis signals in response to DMSO or starvation utilize a group of genes involved in sporulation (spo).     

Starvation and nutrient resuscitation of Klebsiella pneumoniae isolated from oil well waters

Klebsiella pneumoniae isolated from oil well waters reduced in size in response to nutrient starvation. The cells remained viable during starvation and later were able to grow rapidly when stimulated by nutrients. The heterotrophic potential, culture absorbance and extracellular polysaccharide production decreased during cell starvation whereas an initial increase in colony-forming units was observed on agar plates. Transmission electron microscopy (TEM) after 24 d revealed that the cells had changed to small rods or cocci between 0.5 by 0.25 μm and 0.87 by 0.55 μm. When transferred to half-strength brain heart infusion medium, TEM showed cell division and rod-shaped cells after 45 min and full resuscitation within 4 h. Cell response was much slower in sodium citrate medium and resuscitation took 8 h.     

Dark starvation and chloroplast function

Summary Mature spinach plants were held in the dark for several days. The photochemical activities and the activity of some enzymes related to NADP reduction were follwed in the chloroplasts isolated from leaves after dark starvation. Photosystem-II, measured by reduction of DPIP, remained stable during 6 days of darkening. The decrease of NADP reduction which appeared after 2 days of starvation was found to be due to protein autolysis rather than inactivation of the photosystems. The stability of photosystem-I was demonstrated by reactivation of NADP reduction after addition of purified ferredoxin and ferredoxin-NADP-reductase. After 4 days of starvation the restoration of the NADP reduction required in addition another, low-molecular-weight factor. From the isolation procedure and from its properties this factor is assumed to be identical with FRS. However, even in the presence of FRS only half of the total activity is restored after 7 days. The activity of the NADP-reducing system is restored in vivo when plants kept for 7 days in the dark are again illuminated.Abbreviations NADP nicotinamide-adenine-dinucleotide phosphate - DPIP 2,6-dichlorophenolindophenol - DCMU (3,4-dichlorophenyl)-1,1-dimethylurea - FRS ferredoxin-reducing-substance