Starvation induces shrinkage of the bacterial cytoplasm.

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.

The Stringent Response Determines the Ability of a Commensal Bacterium to Survive Starvation and to Persist in the Gut.

In the mammalian gut, bacteria compete for resources to maintain their populations, but the factors determining their success are poorly understood. We report that the human gut bacterium Bacteroides thetaiotaomicron relies on the stringent response, an intracellular signaling pathway that allocates resources away from growth, to survive carbon starvation and persist in the gut. Genome-scale transcriptomics, 13C-labeling, and metabolomics analyses reveal that B. thetaiotaomicron uses the alarmone (p)ppGpp to repress multiple biosynthetic pathways and upregulate tricarboxylic acid (TCA) cycle genes in these conditions. During carbon starvation, (p)ppGpp triggers accumulation of the metabolite alpha-ketoglutarate, which itself acts as a metabolic regulator; alpha-ketoglutarate supplementation restores viability to a (p)ppGpp-deficient strain. These studies uncover how commensal bacteria adapt to the gut by modulating central metabolism and reveal that halting rather than accelerating growth can be a determining factor for membership in the gut microbiome.

Fire frequency drives decadal changes in soil carbon and nitrogen and ecosystem productivity.

Fire frequency is changing globally and is projected to affect the global carbon cycle and climate. However, uncertainty about how ecosystems respond to decadal changes in fire frequency makes it difficult to predict the effects of altered fire regimes on the carbon cycle; for instance, we do not fully understand the long-term effects of fire on soil carbon and nutrient storage, or whether fire-driven nutrient losses limit plant productivity. Here we analyse data from 48 sites in savanna grasslands, broadleaf forests and needleleaf forests spanning up to 65 years, during which time the frequency of fires was altered at each site. We find that frequently burned plots experienced a decline in surface soil carbon and nitrogen that was non-saturating through time, having 36 per cent (±13 per cent) less carbon and 38 per cent (±16 per cent) less nitrogen after 64 years than plots that were protected from fire. Fire-driven carbon and nitrogen losses were substantial in savanna grasslands and broadleaf forests, but not in temperate and boreal needleleaf forests. We also observe comparable soil carbon and nitrogen losses in an independent field dataset and in dynamic model simulations of global vegetation. The model study predicts that the long-term losses of soil nitrogen that result from more frequent burning may in turn decrease the carbon that is sequestered by net primary productivity by about 20 per cent of the total carbon that is emitted from burning biomass over the same period. Furthermore, we estimate that the effects of changes in fire frequency on ecosystem carbon storage may be 30 per cent too low if they do not include multidecadal changes in soil carbon, especially in drier savanna grasslands. Future changes in fire frequency may shift ecosystem carbon storage by changing soil carbon pools and nitrogen limitations on plant growth, altering the carbon sink capacity of frequently burning savanna grasslands and broadleaf forests.

Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism.

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

Understanding of polyhydroxybutyrate production under carbon and phosphorus-limited growth conditions in non-axenic continuous culture.

In a waste into resource strategy, a selection of polyhydroxybutyrate (PHB)-accumulating organisms from activated sludge was achieved in an open continuous culture under acetic acid and phosphorus limitation. Once the microbial population was selected at a dilution rate (D), an increase in phosphorus limitation degree was applied in order to study the intracellular phosphorus plasticity of selected bacteria and the resulting capacity to produce PHB. Whatever D, all selected populations were able to produce PHB. At a D, the phosphorus availability determined the phosphorus-cell content which in turn fixed the amount of cell. All the remaining carbon was thus directed toward PHB. By decreasing D, microorganisms adapted more easily to higher phosphorus limitation leading to higher PHB content. A one-stage continuous reactor operated at D=0.023h(-)(1) gave reliable high PHB productivity with PHB content up to 80%. A two-stage reactor could ensure better productivity while allowing tuning product quality.

Interaction Effects of Light, Temperature and Nutrient Limitations (N, P and Si) on Growth, Stoichiometry and Photosynthetic Parameters of the Cold-Water Diatom Chaetoceros wighamii.

Light (20-450 µmol photons m(-2) s(-1)), temperature (3-11 °C) and inorganic nutrient composition (nutrient replete and N, P and Si limitation) were manipulated to study their combined influence on growth, stoichiometry (C:N:P:Chl a) and primary production of the cold water diatom Chaetoceros wighamii. During exponential growth, the maximum growth rate (~0.8 d(-1)) was observed at high temperature and light; at 3 °C the growth rate was ~30% lower under similar light conditions. The interaction effect of light and temperature were clearly visible from growth and cellular stoichiometry. The average C:N:P molar ratio was 80:13:1 during exponential growth, but the range, due to different light acclimation, was widest at the lowest temperature, reaching very low C:P (~50) and N:P ratios (~8) at low light and temperature. The C:Chl a ratio had also a wider range at the lowest temperature during exponential growth, ranging 16-48 (weight ratio) at 3 °C compared with 17-33 at 11 °C. During exponential growth, there was no clear trend in the Chl a normalized, initial slope (α*) of the photosynthesis-irradiance (PE) curve, but the maximum photosynthetic production (P(m)) was highest for cultures acclimated to the highest light and temperature. During the stationary growth phase, the stoichiometric relationship depended on the limiting nutrient, but with generally increasing C:N:P ratio. The average photosynthetic quotient (PQ) during exponential growth was 1.26 but decreased to <1 under nutrient and light limitation, probably due to photorespiration. The results clearly demonstrate that there are interaction effects between light, temperature and nutrient limitation, and the data suggests greater variability of key parameters at low temperature. Understanding these dynamics will be important for improving models of aquatic primary production and biogeochemical cycles in a warming climate.

Heterologous expression of Anabaena PCC 7120 all3940 (a Dps family gene) protects Escherichia coli from nutrient limitation and abiotic stresses.

This study presents first hand data on the cloning and heterologous expression of Anabaena PCC 7120 all3940 (a dps family gene) in combating nutrients limitation and multiple abiotic stresses. The Escherichia coli transformed with pGEX-5X-2-all3940 construct when subjected to iron, carbon, nitrogen, phosphorus limitation and carbofuron, copper, UV-B, heat, salt and cadmium stress registered significant increase in growth over the cells transformed with empty vector under iron (0%), carbon (0.05%), nitrogen (3.7 mM) and phosphorus (2mM) limitation and carbofuron (0.025 mg ml(-1)), CuCl(2) (1 mM), UV-B (10 min), heat (47 degrees C), NaCl (6% w/v) and CdCl(2) (4mM) stress. Enhanced expression of all3940 gene measured by semi-quantitative RT-PCR at different time points under above mentioned treatments clearly demonstrates its role in tolerance against aforesaid abiotic stresses. This study opens the gate for developing transgenic cyanobacteria capable of growing successfully under above mentioned stresses.

Glucose upshift of carbon-starved marine Vibrio sp. strain S14 causes amino acid starvation and induction of the stringent response.

The physiological status of carbon-starved cells of the marine Vibrio sp. strain S14 has been investigated by the analysis of their immediate response to carbon and energy sources. During the first minute after glucose addition to 48-h-starved cells, the pools of ATP and GTP increased rapidly, and the [ATP]/[ADP] ratio reached the level typical for growing cells within 4 min. The total rates of RNA and protein synthesis increased initially but were inhibited 4 to 5 min after glucose addition by the induction of the stringent response. A mutation in the relA gene abolished stringent control during the recovery and significantly prolonged the lag phase, before the starved cells regrew, after the addition of a single source of carbon. However, both the wild-type and the relA cells regrew without a significant lag phase when given glucose supplemented with amino acids. On the basis of these results, it is suggested that carbon-starved cells are deficient in amino acid biosynthesis and that ppGpp and the stringent response are involved in overcoming this deficiency, presumably by depressing the synthesis of amino acid biosynthetic enzymes. Furthermore, the data suggest that the starved cells primarily are starved for energy, and evidence is presented that the step-up in the rate of protein synthesis after refeeding is partially dependent on de novo RNA synthesis.

In vitro characterization of a phosphate starvation-independent carbon-phosphorus bond cleavage activity in Pseudomonas fluorescens 23F.

A novel, metal-dependent, carbon-phosphorus bond cleavage activity, provisionally named phosphonoacetate hydrolase, was detected in crude extracts of Pseudomonas fluorescens 23F, an environmental isolate able to utilize phosphonoacetate as the sole carbon and phosphorus source. The activity showed unique specificity toward this substrate; its organic product, acetate, was apparently metabolized by the glyoxylate cycle enzymes of the host cell. Unlike phosphonatase, which was also detected in crude extracts of P. fluorescens 23F, phosphonoacetate hydrolase was inducible only in the presence of its sole substrate and did not require phosphate starvation.

[Effect of a deficiency of carbon, nitrogen, phosphorus and magnesium in the growth medium on the mechanical properties of Escherichia coli cell walls]. / Vliianie defitsita ugleroda, azota, fosfora i magniia v srede vyrashchivaniia na mekhanicheskie svoistva kletochnykh stenok Escherichia coli.

Values of modulus of elasticity of cell walls and strength level of cells Escherichia coli cultivated in the carbon, nitrogen and phosphorus deficient media or incubated in the magnesium-free medium were determined. Elastic modulus of cells grown in the magnesium-free medium was by two order of magnitude lower than that of the control cells. Elastic modulus of cells cultivated in the nitrogen and carbon deficient media was by one and two orders of magnitude lower than in the control cells whereas strength level was by 1.15 and 1.39 times higher, respectively. Elastic modulus of cells grown in the phosphorus deficient medium remained undetermined and strength level of those cells proved to be the lowest (0.9 of the control).