Gene expression in response to multiple nutrient-starvation conditions in Salmonella typhimurium

Abstract In nature, bacteria encounter a variety of environmental conditions, among the most frequent of these is the limitation or starvation of one or more essential nutrients. It is reasonable to assume, therefore, that bacteria have evolved mechanisms to enhance their survival over prolonged periods of nutrient starvation. We have identified eight genetic loci in the enteropathogen Salmonella typhimurium , using Mu d -directed lacZ operon fusion technology, that were induced in response to two or more different starvation conditions (sti) . In simultaneous studies, we also identified genetic loci, using Mu d-lac fusions, which respond to only phosphate-starvation conditions (psi) . We further characterized these loci as to their induction-characteristics, kinetics of induction, expression during growth on different carbon sources, and approximate location on the S. typhimurium genetic map. In concurrent studies, we analyzed whole cell extracts of S. typhimurium grown under a variety of nutrients oxydation conditions as well as under nonlimiting conditions, using two-dimensional polyacrylamide gel electrophoresis. Results from these studies correlated well with our gene fusion studies. In more recent studies, we have demonstrated a complex genetic regulation of a number of these starvation-inducible loci, and have implicated at least four of these loci in the long term starvation-survival of S. typhimurium .     

The alternative sigma factor sigmaE controls antioxidant defences required for Salmonella virulence and stationary-phase survival

Bacteria must contend with conditions of nutrient limitation in all natural environments. Complex programmes of gene expression, controlled in part by the alternative sigma factors sigmaS (sigma38, RpoS) and sigmaH (sigma32, RpoH), allow a number of bacterial species to survive conditions of partial or complete starvation. We show here that the alternative sigma factor sigmaE (sigma24, RpoE) also facilitates the survival of Salmonella typhimurium under conditions of nutrient deprivation. Expression of the sigmaE regulon is strongly induced upon entry of Salmonella into stationary phase. A Salmonella mutant lacking sigmaE has reduced survival during stationary phase as well as increased susceptibility to oxidative stress. A Salmonella strain lacking both sigmaE and sigmaS is non-viable after just 24 h in stationary phase, but survival of these mutants is completely preserved under anaerobic stationary-phase conditions, suggesting that oxidative injury is one of the major mechanisms of reduced microbial viability during periods of nutrient deprivation. Moreover, the attenuated virulence of sigmaE-deficient Salmonella for mice can be largely restored by genetic abrogation of the host phagocyte respiratory burst, suggesting that the sigmaE regulon plays an important antioxidant role during Salmonella infection of mammalian hosts.     

Mobilization of vacuolar arginine in Neurospora crassa. Mechanism and role of glutamine

Nitrogen starvation has been shown to increase the cytosolic arginine concentration and to accelerate protein turnover in mycelia of Neurospora crassa. The cytosolic arginine is derived from a metabolically inactive vacuolar pool. Redistribution of arginine between cytosolic and vacuolar compartments is the result of mobilization of this metabolite in response to nitrogen starvation. Mobilization of arginine (and purines) also occurred in response to glutamine limitation, but arginine accumulated upon proline starvation. These observations indicate that mobilization is a consequence of glutamine limitation rather than a general response to amino acid starvation (or limitation). Analysis of the amino acid pools in mycelia subjected to starvation or limitation suggests that glutamine (or a metabolite derived from glutamine) provides a signal which determines the metabolic fate of vacuolar arginine. The results are consistent with the hypothesis that vacuolar compartmentation provides a readily available store of nitrogen-rich compounds to be utilized during differentiation or under conditions of nutritional stress.     

Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation.

The amounts of glycogen and trehalose have been measured in cells of a prototrophic diploid yeast strain subjected to a variety of nutrient limitations. Both glycogen and trehalose were accumulated in cells deprived specifically of nirogen, sulfur, or phosphorus, suggesting that reserve carbohydrate accumulation is a general response to nutrient limitation. The patterns of accumulation and utilization of glycogen and trehalose were not identical under these conditions, suggesting that the two carbohydrates may play distinct physiological roles. Glycogen and trehalose were also accumulated by cells undergoing carbon and energy limitation, both during diauxic growth in a relatively poor medium and during the approach to stationary phase in a rich medium. Growth in the rich medium was shown to be carbon or energy limited or both, although the interaction between carbon source limitation and oxygen limitation was complex. In both media, the pattern of glycogen accumulation and utilization was compatible with its serving as a source of energy both during respiratory adaptation and during a subsequent starvation. In contrast, the pattern of trehalose accumulation and utilization seemed compatible only with the latter role. In cultures that were depleting their supplies of exogenous glucose, the accumulation of glycogen began at glucose concentrations well above those sufficient to suppress glycogen accumulation in cultures growing with a constant concentration of exogenous glucose. The mechanism of this effect is not clear, but may involve a response to the rapid rate of change in the glucose concentration.     

AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana

Vacuolar autophagy is a major pathway by which eukaryotic cells degrade macromolecules, either to remove damaged or unnecessary proteins, or to produce respiratory substrates and raw materials to survive periods of nutrient deficiency. During autophagy, a double membrane forms around cytoplasmic components to generate an autophagosome, which is transported to the vacuole. The outer membrane fuses with the vacuole or lysosome, and the inner membrane and its contents are degraded by vacuolar or lysosomal hydrolases. We have identified a small gene family in Arabidopsis thaliana, members of which show sequence similarity to the yeast autophagy gene ATG18. Members of the AtATG18 gene family are differentially expressed in response to different growth conditions, and one member of this family, AtATG18a, is induced both during sucrose and nitrogen starvation and during senescence. RNA interference was used to generate transgenic lines with reduced AtATG18a expression. These lines show hypersensitivity to sucrose and nitrogen starvation and premature senescence, both during natural senescence of leaves and in a detached leaf assay. Staining with the autophagosome-specific fluorescent dye monodansylcadaverine revealed that, unlike wild-type plants, AtATG18a RNA interference plants are unable to produce autophagosomes in response to starvation or senescence conditions. We conclude that the AtATG18a protein is likely to be required for autophagosome formation in Arabidopsis.     

Starvation tolerance associated with prolonged sleep bouts upon starvation in a single natural population of Drosophila melanogaster

Large genetic variations in starvation tolerance in animals indicate that there are multiple strategies to cope with low‐nutrient conditions. Fruit flies (Drosophila melanogaster) typically respond to starvation by suppressing sleep and enhancing locomotor activity presumably to search for food. However, we hypothesized that in a natural population, there are costs and benefits to sleep suppression under low‐nutrient conditions and that conserving energy through sleep could be a better strategy depending on food availability. In this study, we quantified the variation in sleep‐related traits in 21 wild‐derived inbred lines from Katsunuma, Japan, under fed and starved conditions and analysed the relationship between those traits and starvation tolerance. Although most of the lines responded to starvation by suppressing the total time in sleep, there were indeed two lines that responded by significantly increasing the sleep‐bout durations and thus not reducing the total time in sleep. These genotypes survived longer in acute starvation conditions compared to genotypes that responded by the immediate suppression of sleep, which could be due to the reduced metabolic rate during the long uninterrupted sleep bouts. The coexistence of the enhanced foraging and resting strategies upon starvation within a single population is consistent with the presence of a behavioural trade‐off between food search and energy conservation due to unpredictable food availability in nature. These results provide insights into the evolutionary mechanisms that contribute to the maintenance of genetic variations underlying environmental stress resistance.     

Profiling the Bordetella pertussis proteome during iron starvation

Regulation of gene expression in response to local iron concentration is commonly observed in bacterial pathogens that face this nutrient limitation during host infection. In this study, a proteomic approach was used to analyze the differential protein expression of Bordetella pertussis under iron limitation. Whole cell lysates (WCL) and outer membrane fractions of bacteria grown either under iron-starvation or iron-excess conditions were analyzed by two-dimensional (2-D) gel electrophoresis. Statistical analysis revealed 36 proteins displaying differential expression, 9 with higher expression under iron-excess and 27 with increased expression under iron-starvation. These proteins were subjected to tryptic digestion and MALDI-TOF MS. Apart from those previously reported, we identified new low-iron-induced proteins that might help to explain the increased virulence of this phenotype. Additionally, we found evidence that at least one of the identified proteins, solely expressed under iron starvation, is highly immunogenic in infected individuals.     

Signatures of nitrogen limitation in the elemental composition of the proteins involved in the metabolic apparatus

Nitrogen (N) is a fundamental component of nucleotides and amino acids and is often a limiting nutrient in natural ecosystems. Thus, study of the N content of biomolecules may establish important connections between ecology and genomics. However, while significant differences in the elemental composition of whole organisms are well documented, how the flux of nutrients in the cell has shaped the evolution of different cellular processes remains poorly understood. By examining the elemental composition of major functional classes of proteins in four multicellular eukaryotic model organisms, we find that the catabolic machinery shows substantially lower N content than the anabolic machinery and the rest of the proteome. This pattern suggests that ecological selection for N conservation specifically targets cellular components that are highly expressed in response to nutrient limitation. We propose that the RNA component of the anabolic machineries is the mechanistic force driving the elemental imbalance we found, and that RNA functions as an intracellular nutrient reservoir that is degraded and recycled during starvation periods. A comparison of the elemental composition of the anabolic and catabolic machineries in species that have experienced different levels of N limitation in their evolutionary history (animals versus plants) suggests that selection for N conservation has preferentially targeted the catabolic machineries of plants, resulting in a lower N content of the proteins involved in their catabolic processes. These findings link the composition of major cellular components to the environmental factors that trigger the activation of those components, suggesting that resource availability has constrained the atomic composition and the molecular architecture of the biotic processes that enable cells to respond to reduced nutrient availability.     

Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type‐2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter

When cultivated under stress conditions, many plants and algae accumulate oil. The unicellular green microalga Chlamydomonas reinhardtii accumulates neutral lipids (triacylglycerols; TAGs) during nutrient stress conditions. Temporal changes in TAG levels in nitrogen (N)‐ and phosphorus (P)‐starved cells were examined to compare the effects of nutrient depletion on TAG accumulation in C. reinhardtii. TAG accumulation and fatty acid composition were substantially changed depending on the cultivation stage before nutrient starvation. Profiles of TAG accumulation also differed between N and P starvation. Logarithmic‐growth‐phase cells diluted into fresh medium showed substantial TAG accumulation with both N and P deprivation. N deprivation induced formation of oil droplets concomitant with the breakdown of thylakoid membranes. In contrast, P deprivation substantially induced accumulation of oil droplets in the cytosol and maintaining thylakoid membranes. As a consequence, P limitation accumulated more TAG both per cell and per culture medium under these conditions. To enhance oil accumulation under P deprivation, we constructed a P deprivation‐dependent overexpressor of a Chlamydomonas type‐2 diacylglycerol acyl‐CoA acyltransferase (DGTT4) using a sulphoquinovosyldiacylglycerol 2 (SQD2) promoter, which was up‐regulated during P starvation. The transformant strongly enhanced TAG accumulation with a slight increase in 18 : 1 content, which is a preferred substrate of DGTT4. These results demonstrated enhanced TAG accumulation using a P starvation–inducible promoter.     

Accumulation of ppGpp and ppGp in Staphylococcus aureus 8325-4 following nutrient starvation

AIM: To investigate the accumulation of highly phosphorylated guanosine nucleotides in Staphylococcus aureus 8325-4 following nutrient deprivation. METHODS AND RESULTS: Nutrient shiftdown of Staph. aureus, HPLC of nucleotides and Western blotting of cell-free extracts. ppGpp rapidly accumulated when cells were deprived of isoleucine following addition of mupirocin, or after carbon deprivation. In contrast, total amino acid starvation led to delayed production of ppGp, which suggests that Staph. aureus exhibits a unique response to total amino acid deprivation compared with other eubacteria. Intracellular ppGp was observed at high levels under all starvation conditions, which suggests that this nucleotide is linked to nutrient limitation and may therefore be involved in regulating the stringent response in Staph. aureus. pppGpp was not observed under any nutrient-limiting condition. Western blot analysis of whole-cell extracts from Staph. aureus 8325-4, showed that antibodies to RelA and SpoT cross-reacted under conditions that detected these proteins in Escherichia coli. CONCLUSIONS: Staph. aureus produces ppGpp and ppGp following nutrient limitation. Immunological analysis indicates that Staph. aureus contains RelA and SpoT proteins, similar to those produced by E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a new example of the diversity of metabolic regulations in bacteria.     

G‐protein coupled receptor‐mediated nutrient sensing and developmental control in Aspergillus nidulans

Nutrient sensing and utilisation are fundamental for all life forms. As heterotrophs, fungi have evolved a diverse range of mechanisms for sensing and taking up various nutrients. Despite its importance, only a limited number of nutrient receptors and their corresponding ligands have been identified in fungi. G‐protein coupled receptors (GPCRs) are the largest family of transmembrane receptors. The Aspergillus nidulans genome encodes 16 putative GPCRs, but only a few have been functionally characterised. Our previous study showed the increased expression of an uncharacterised putative GPCR, gprH, during carbon starvation. GprH appears conserved throughout numerous filamentous fungi. Here, we reveal that GprH is a putative receptor involved in glucose and tryptophan sensing. The absence of GprH results in a reduction in cAMP levels and PKA activity upon adding glucose or tryptophan to starved cells. GprH is pre‐formed in conidia and is increasingly active during carbon starvation, where it plays a role in glucose uptake and the recovery of hyphal growth. GprH also represses sexual development under conditions favouring sexual fruiting and during carbon starvation in submerged cultures. In summary, the GprH nutrient‐sensing system functions upstream of the cAMP‐PKA pathway, influences primary metabolism and hyphal growth, while represses sexual development in A. nidulans.     

Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation

Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.     

Aspartokinase II from Bacillus subtilis is degraded in response to nutrient limitation

Aspartokinase II from Bacillus subtilis was shown by immunochemical methods to be regulated by degradation in response to starvation of cells for various nutrients. Ammonium starvation induced the fastest aspartokinase II decline (t1/2 = 65 min), followed by amino acid starvation (t1/2 = 80 min) and glucose limitation (t1/2 = 120 min). Loss of enzyme activity was closely correlated with the disappearance of the alpha subunit; degradation of the beta subunit was somewhat delayed or slower under some conditions. Pulse-chase experiments demonstrated that aspartokinase II was stable during exponential growth; the synthesis of the enzyme rapidly declined in response to nutrient exhaustion. The degradation of aspartokinase II was interrupted by inhibitors of energy production and protein synthesis but was not changed in a mutant lacking a major intracellular protease. Mutants lacking a normal stringent response displayed only a slight decrease in the rate of aspartokinase II degradation, even though aspartate transcarbamylase was degraded more slowly in the same mutant cells. These results indicate that although energy-dependent degradation of biosynthetic enzymes is a general phenomenon in nutrient-starved B. subtilis cells, the degradation of specific enzymes probably involves different pathways.     

RpoS is necessary for both the positive and negative regulation of starvation survival genes during phosphate, carbon, and nitrogen starvation in Salmonella typhimurium.

The starvation stress response of Salmonella typhimurium encompasses the genetic and physiologic changes that occur when this bacterium is starved for an essential nutrient such as phosphate (P), carbon (C), or nitrogen (N). The responses to the limitation of each of these nutrients involve both unique and overlapping sets of proteins important for starvation survival and virulence. The role of the alternative sigma factor RpoS in the regulation of the starvation survival loci, stiA, stiB, and stiC, has been characterized. RpoS (sigma S) was found to be required for the P, C, and N starvation induction of stiA and stiC. In contrast, RpoS was found to be required for the negative regulation of stiB during P and C starvation-induced stationary phase but not during logarithmic phase. This role was independent of the relA gene (previously found to be needed for stiB induction). The role of RpoS alone and in combination with one or more sti mutations in the starvation survival of the organism was also investigated. The results clearly demonstrate that RpoS is an integral component of the complex interconnected regulatory systems involved in S. typhimurium's response to nutrient deprivation. However, differential responses of various sti genes indicate that additional signals and regulatory proteins are also involved.