Stress-induced proteins of Agrobacterium tumefaciens

The pattern of proteins produced by bacteria represents the physiological state of the organism as well as the environmental conditions encountered. Environmental stress induces the expression of several regulons encoding stress proteins. Extensive information about the proteins which constitute these regulons (or stimulons) and their control is available for very few bacteria, such as the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli (gamma-proteobacteria) and is minimal for all other bacteria. Agrobacterium tumefaciens is a Gram-negative plant pathogen of the alpha-proteobacteria, which constitutes the main tool for plant recombinant genetics. Our previous studies on the control of chaperone-coding operons indicated that A. tumefaciens has unique features and combines regulatory elements from both B. subtilis and E. coli. Therefore, we examined the patterns of proteins induced in A. tumefaciens by environmental changes using two-dimensional gel electrophoresis and dual-channel image analysis. Shifts to high temperature, oxidative and mild acid stresses stimulated the expression of 97 proteins. The results indicate that most of these stress-induced proteins (80/97) were specific to one stress stimulon. Only 10 proteins appear to belong to a general stress regulon.     

General stress sigma factor RpoS influences time required to enter the viable but non-culturable state in Salmonella enterica

In stressful conditions, bacteria enter into the viable but non-culturable (VBNC) state; in this state, they are alive but fail to grow on conventional media on which they normally grow and develop into colonies. The molecular basis underlying this state is unknown. We investigated the role of the alternative sigma factor RpoS (σ(38)) in the VBNC induction using Salmonella Dublin, Salmonella Oranienburg and Salmonella Typhimurium LT2. VBNC was induced by osmotic stress in LT2 and Oranienburg. Dublin also entered the VBNC state, but more slowly than LT2 and Oranienburg did. The LT2 rpoS gene was initiated from an alternative initiation codon, TTG; therefore, LT2 had smaller amounts of RpoS than Dublin and Oranienburg. Oranienburg had a single amino acid substitution (D118N) in RpoS (RpoS(SO)). Disruption of rpoS caused rapid VBNC induction. VBNC induction was significantly delayed by Dublin-type RpoS (RpoS(SD)), but only slightly by RpoS(SO). These results indicate that RpoS delays VBNC induction and that the rapid induction of VBNC in LT2 and Oranienburg may be due to lower levels of RpoS and to the D118N amino acid substitution, respectively. Reduced RpoS intracellular level was observed during VBNC induction. During the VBNC induction, Salmonella might regulate RpoS which is important for maintenance of culturablity under stresses.     

Differences in strategies to combat osmotic stress in Burkholderia cenocepacia elucidated by NMR-based metabolic profiling

Aims: To investigate mechanisms of osmotic tolerance in Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc) of closely related strains, which is of clinical as well as environmental importance. Methods and Results: We employed NMR‐based metabolic profiling (metabolomics) to elucidate the metabolic consequences of high osmotic stress for five isolates of B. cenocepacia. The strains differed significantly in their levels of osmotic stress tolerance, and we identified three different sets of metabolic responses with the strains least impacted by osmotic stress exhibiting higher levels of the osmo‐protective metabolites glycine‐betaine and/or trehalose. Strains either increased concentrations or had constitutively high levels of these metabolites. Conclusions: Even within the small set of B. cenocepacia isolates, there was a surprising degree of variability in the metabolic responses to osmotic stress. Significance and impact of the study: The metabolic responses, and hence osmotic stress tolerance, vary between different B. cenocepacia isolates. This study provides a first look into the potentially highly diverse physiology of closely related strains of one species of the Bcc and illustrates that physiological or clinically relevant phenotypes are unlikely to be inferable from genetic relatedness within this species group.     

Differential genetic interactions of yeast stress response MAPK pathways

Genetic interaction screens have been applied with great success in several organisms to study gene function and the genetic architecture of the cell. However, most studies have been performed under optimal growth conditions even though many functional interactions are known to occur under specific cellular conditions. In this study, we have performed a large‐scale genetic interaction analysis in Saccharomyces cerevisiae involving approximately 49 × 1,200 double mutants in the presence of five different stress conditions, including osmotic, oxidative and cell wall‐altering stresses. This resulted in the generation of a differential E‐MAP (or dE‐MAP) comprising over 250,000 measurements of conditional interactions. We found an extensive number of conditional genetic interactions that recapitulate known stress‐specific functional associations. Furthermore, we have also uncovered previously unrecognized roles involving the phosphatase regulator Bud14, the histone methylation complex COMPASS and membrane trafficking complexes in modulating the cell wall integrity pathway. Finally, the osmotic stress differential genetic interactions showed enrichment for genes coding for proteins with conditional changes in phosphorylation but not for genes with conditional changes in gene expression. This suggests that conditional genetic interactions are a powerful tool to dissect the functional importance of the different response mechanisms of the cell.     

NMR analysis of metabolic responses to extreme conditions of the temperature-dependent coral pathogen Vibrio coralliilyticus

Aims: To identify and understand the presence of metabolites responsible for the variation in the metabolic profile of Vibrio coralliilyticus under extreme conditions. Methods and Results: Multiple batches of V. coralliilyticus were grown under normal conditions. Four samples in one batch were subjected to extreme conditions via a freeze‐thaw cycle during lyophilization. Polar metabolites were extracted using a combination of methanol, water and heat. Nuclear magnetic resonance (NMR)‐based metabolic profiles indicated significant differences between the normal and stressed samples. Three compounds identified in the stressed metabolome were maltose, ethanolamine, and the bioplastic‐type compound (BTC) 2‐butenoic acid, 2‐carboxy‐1‐methylethyl ester. This is the first report of the production of this BTC by V. coralliilyticus. Conclusions: The presence of maltose and ethanolamine indicates a state of acute nutrient limitation; therefore, we hypothesize that the cell’s metabolism turned to its own cell wall, or perhaps neighbouring cells, for sources of carbon and nitrogen. The presence of the BTC also supports the acute nutrient limitation idea because of the parallels with polyhydroxyalkanoate (PHA) production in other gram‐negative bacteria, including other Vibrio species. Significance and Impact of the Study: Recent metabolomics research on the temperature‐dependent coral pathogen V. coralliilyticus has led to the discovery of several compounds produced by the organism as a response to high density, low nutrient conditions. The three metabolites, along with ¹H NMR metabolic fingerprints of the nutrient limited samples, are proposed to serve as metabolic markers for extremely stressful conditions of V. coralliilyticus.     

Adaptation versus shock response to polyethylene glycol-induced low water potential in cultured potato cells

We compared long-term adaptation versus short-term or shock response of potato ( Solanum tuberosum ) cells to polyethylene glycol (PEG)-induced low water potential. Potato cells, which were allowed to adapt gradually to a decreasing water potential, were able to grow actively in a medium containing 20% PEG. In contrast, no appreciable gain in dry weight was observed in potato cells shocked by abrupt transfer to the same medium. PEG-adapted cells were also salt-tolerant, as they were able to proliferate in a medium supplemented with 200 m M NaCl. No visible ultrastructural changes of mitochondria or proplastids were observed in adapted cells at values of low water potential (about −2.0 MPa), which caused membrane disruption and appearance of lipid droplets in unadapted cells. ABA cellular content increased 5-fold in PEG-shocked cells but no significant increase was found in PEG-adapted cells. The intracellular content of free proline increased 12.5 times over the basal level in PEG-adapted cells and 6.5 times in PEG-shocked cells. As shown by in vivo protein labeling, shock conditions strongly inhibited protein synthesis, which was completely recovered in PEG-adapted cells. Osmotin, a protein associated with salt adaptation in tobacco, was constitutively expressed at a high level in PEG-adapted cells and accumulated in PEG-shocked cells only three days after the transfer in a medium supplemented with 20% PEG. Proline and osmotin accumulation were coincident with the increase in cellular ABA content in PEG-shocked cells, but not in PEG-adapted cells. These data suggest that this hormone is mainly involved in shock response rather than long-term adaptation.     

Adaptive characteristics of salt-induced myceloids of Arthrobacter globiformis

When Arthrobacter globiformis is grown in medium containing increased concentrations of NaCl or decreased levels of cations, the bacteria grow as clusters of branching myceloid cells. The sensitivities of salt-induced and citrate-induced myceloids to several environmental stresses were compared to those of normal exponential-phase bacilli and stationary-phase cocci. Salt-induced myceloids were more resistant than normal cells to ultraviolet light or heat shock at 45°C but not to osmotic upshock or pH 4.3; citrate-induced myceloids showed an intermediate rate of heat inactivation. Carbon or nitrogen starvation of myceloids in the absence of added NaCl or citrate led to their division into single cells. Both myceloids and the single cells derived from them were more resistant than normal bacteria to nitrogen starvation. Salt-induced and citrate-induced myceloids showed reduced metabolism of many different carbon compounds in Biolog GP plates. These studies suggest that the formation of multicellular structures by A. globiformis is an adaptive response which increases its potential for survival.     

Swimming performance and metabolic rate of three tropical fishes in relation to temperature

Oxygen consumption was measured for three tropical fishes,Exodon paradoxus, Leporinus fasciatus andLabeo erythrurus in relation to swimming speed and temperature. For each species the logarithm of oxygen consumption (mg 0₂ · g^–1 · h^–1) increased linearly with relative swimming speed (1 · s^–1) with the value of the regression coefficients varying inversely with temperature. Active metabolism and critical swimming speed ofE. paradoxus andL. fasciatus increased with temperature to a maximum at 30 and 35° C respectively. Basal metabolic rates ofE. paradoxus andL. fasciatus increased with temperature. Metabolic rates and critical swimming speed of the three fishes studied were consistent with values for polar, temperate and other tropical species over their respective thermal ranges of tolerance. Tropical fishes have lowered their metabolism and swimming performance from that expected for many temperate species at the same temperature.     

Temporal changes in stress and tissue-specific metabolic responses to municipal wastewater effluent exposure in rainbow trout

Sub-chronic exposure to municipal wastewater effluent (MWWE) in situ was recently shown to impact the acute response to a secondary stressor in rainbow trout (Oncorhynchus mykiss). However, little is known about whether MWWE exposure in itself is stressful to the animal. To address this, we carried out a laboratory study to examine the organismal and cellular stress responses and tissue-specific metabolic capacity in trout exposed to MWWE. Juvenile rainbow trout were exposed to 0, 20 and 90% MWWE (from a tertiary wastewater treatment plant), that was replenished every 2d, for 14 d. Fish were sampled 2, 8 or 14 d post-exposure. Plasma cortisol, glucose and lactate levels were measured as indicators of organismal stress response, while inducible heat shock protein 70 (hsp70), constitutive heat shock protein 70 (hsc70) and hsp90 expression in the liver were used as markers of cellular stress response. Impact of MWWE on cortisol signaling was ascertained by determining glucocorticoid receptor protein (GR) expression in the liver, brain and, heart, and metabolic capacity was evaluated by measuring liver glycogen content and tissue-specific activities of key enzymes in intermediary metabolism. Plasma glucose and lactate levels were unaffected by exposure to MWWEs, whereas cortisol showed a transient increase in the 20% group at 8d. Liver hsc70 and hsp90, but not hsp70 expression, were higher in the 90% MWWE group after 8d. There was a temporal change in GR expression in the liver and heart, but not brain of trout exposed to MWWE. Liver glycogen content and activities liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and alanine aminotransferase (AlaAT) were significantly affected by MWWE exposure. The glycolytic enzymes pyruvate kinase (PK) and hexokinase (HK) activities were significantly higher temporally by MWWE exposure in the gill and heart, but not in the liver and brain. Overall, a 14 d exposure to MWWE evokes a cellular stress response and perturbs the cortisol stress response in rainbow trout. The tissue-specific temporal changes in the metabolic capacity suggest enhanced energy demand in fish exposed to MWWE, which may eventually lead to reduced fitness.     

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.     

Seasonal changes in growth and standard metabolic rate of juvenile perch, Perca fluviatilis L.

The maximum growth rate of juvenile perch, PercaJuviatilis L., at different constant temperatures and in naturally changing day-lengths was studied in the laboratory. Standard metabolic rate was studied in starvation experiments at constant temperatures under short- and long-day conditions. Growth occurred in temperatures above 8 to 10°C. In winter, from mid-October until mid-April, maximal growth was considerably reduced and was relatively slow but constant. The standard metabolic rate was reduced c . 50% under short-day conditions. The seasonal change in metabolic rates, presumably controlled by an endogenous rhythm, was considered to be an adaptation to low food availability during the short winter days.     

Stress-induced nuclear condensation of NELF drives transcriptional downregulation

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Protein catabolism in growing Bacillus megaterium during adaptation to salt stress

Elevated concentration of NaCl in liquid medium caused a concentration-dependent growth delay (adaptation lag) and decrease in the maximal growth rate of Bacillus megaterium. The adaptation to salt stress was accompanied by transformation of some otherwise stable (long-lived; LLP) cell proteins into quickly degraded (short-lived; SLP) ones. Exposure to the strongly growth-reducing 1 M NaCl increased the size of the SLP 'pool' of intracellular proteins from about 5 to about 15% of total protein. The major intracellular proteolytic capacity of B. megaterium is represented by intracellular serine proteinases (ISP). Paradoxically, their specific activity was lowered or masked during the adaptation phase marked by increased catabolism of short-lived and/or destabilized proteins by the stress. This documents that intracellular proteolytic activity cannot be a key regulator of protein catabolism during adaptation to stress.     

Bacterial persistence is an active σS stress response to metabolic flux limitation

While persisters are a health threat due to their transient antibiotic tolerance, little is known about their phenotype and what actually causes persistence. Using a new method for persister generation and high‐throughput methods, we comprehensively mapped the molecular phenotype of Escherichia coli during the entry and in the state of persistence in nutrient‐rich conditions. The persister proteome is characterized by σ^S‐mediated stress response and a shift to catabolism, a proteome that starved cells tried to but could not reach due to absence of a carbon and energy source. Metabolism of persisters is geared toward energy production, with depleted metabolite pools. We developed and experimentally verified a model, in which persistence is established through a system‐level feedback: Strong perturbations of metabolic homeostasis cause metabolic fluxes to collapse, prohibiting adjustments toward restoring homeostasis. This vicious cycle is stabilized and modulated by high ppGpp levels, toxin/anti‐toxin systems, and the σ^S‐mediated stress response. Our system‐level model consistently integrates past findings with our new data, thereby providing an important basis for future research on persisters.     

Differences in adaptive stabilization of structures in response to stress and hypoxia relate with the accumulation of hsp70 isoforms

The phenomenon of adaptive stabilization of structures (PhASS) develops during adaptation of the organism to intermittent restraint stress. The PhASS manifests itself in a considerably increased resistance of the heart to a broad spectrum of harmful factors. In the present work, the content of hsp70 and their role in the development of PhASS during adaptation to intermittent restraint stress and to intermittent hypoxia were studied. In adaptation to restraint stress, five hsp70 isoforms with pI ranging from 5.7 to 6.3 were accumulated in the myocardium. The heart simultaneously became strikingly resistant to reperfusion paradox and heat shock. In adaptation to hypoxia, only two hsp70 isoforms with pI about 5.8 were accumulated. The resistance to reperfusion paradox was not increased and the resistance to heat shock was increased only moderately. These data suggest a role of different hsp70 isoforms in the mechanism of PhASS as well as adaptive protection of the heart.Abbreviation hsp70 heat shock proteins - PhASS Phenomenon of Adaptive Stabilization of Structures - CK Creatine Kinase     

Response of seedlings of radish (Raphanus sativus) to osmotic shock and external hydrostatic pressure

The effects of an osmotic shock (300 m M mannitol, –0. 67 MPa) or/and increased external hydrostatic pressure on seedlings (42-h-germinated seeds) of radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) were investigated. The osmotic shock did not inhibit H⁺ extrusion and net K^- uptake, and did not affect the levels of malic acid, reducing sugars, sucrose or amino acids or of the energy charge (i. e. the synthesis of energy-rich phosphate bonds), but inhibited the synthesis of proteins. RNA and DNA, measured as incorporation of labelled precursors. When the osmotic shock was applied together with an increased external hydrostatic pressure of the same magnitude (+0. 67 MPa), the same metabolic parameters and the inhibition of synthesis of RNA and DNA were not substantially affected, while the inhibition of protein synthesis was practically reversed and the energy charge decreased; the recovery of protein synthesis was not due to a change in labelled leucine uptake capability. Increased external hydrostatic pressure alone decreased the energy charge without affecting the other parameters considered.
The possibility that protein synthesis activity is directly controlled by cell turgor pressure (internal hydrostatic pressure) is discussed.