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Influence of growth rate and nutrient limitation on the gross cellular composition of Pseudomonas aeruginosa and its resistance to 3- and 4-chlorophenol.

Concentrations of 3-chlorophenol and 4-chlorophenol below their minimal inhibitory concentrations were found to increase the permeability of Pseudomonas aeruginosa cells to protons. Levels of such activity were assessed in suspensions of cells prepared from chemostat-grown cultures, limited by either magnesium (Mg-lim) or glucose (G-lim), with the use of five growth rates. Drug concentrations required to produce the same levels of proton translocation varied with the growth rate and the nutrient limiting growth. Fast-growing cultures were more sensitive than slower-growing ones, and G-lim cells were generally more sensitive than Mg-lim ones. 3-Chlorophenol had greater activity than 4-chlorophenol at slow growth rates, but at faster rates of growth their activity was similar. Variation in these iso-effective concentrations for different cells probably reflected an alteration in the ease of drug penetration of the outer envelope. Uptake of the compounds by cells in suspension varied, drug-sensitive bacteria absorbing more than resistant ones. This variation in uptake persisted when bacteria were solvent-extracted to remove readily extractable lipids (REL). Since no significant alteration in cell size was observed among the growth conditions studied, variation in absorption probably resulted from an altered affinity of the cells to the drug, with little involvement of REL. Overall REL content did not alter significantly with growth rate or nutrient limitation. However, total phospholipid content decreased and fatty acid content increased with increasing growth rate. For G-lim and Mg-lim cultures phosphatidylcholine content remained constant, yet phosphatidylethanolamine and phosphatidylglycerol content decreased with increasing growth rate. Diphosphatidylglycerol content decreased with increasing growth rate for Mg-lim cultures and remained relatively constant for G-lim cultures. Lipopolysaccharide content of the cells was higher in Mg-lim than in G-lim cultures and decreased with increasing growth rate in both cases. Lipopolysaccharide content correlated significantly with drug uptake and sensitivity, and it appeared to determine the degree of penetration of the cell envelope by these chlorinated phenols.

Chlorophenols

Growth and sporulation of auxotrophs of Bacillus subtilis in a medium with limited nutrients.

Growth and sporulation were examined for 30 auxotrophs of Bacillus subtilis in a chemically defined medium with suboptimal amounts of nutrients. All strains except for some adenine-requiring mutants could not overtake sporulation stage II when amino acids, vitamins, or bases were limited, whereas they sporulated fairly well without limitation. Abnormal structures, a cell with thickened cell wall and a cell with several refractile bodies, were found in some strains after the vegetative growth stopped.

Adenine

Global microbial DNA signatures of temperature and nutrient limitation across ecosystems.

Microbial genomes continuously adapt to environmental conditions, but identifying universal signatures of adaptation remains challenging. Here we show that environmental temperature can be accurately predicted across ecosystems from DNA composition alone (R2 = 0.75), using tetranucleotide frequencies from 1,235 marine and soil metagenomes and a machine learning approach. This predictive signal was also apparent within individual taxa, consistent with a fundamental temperature-associated signature. By contrast, GC content exhibited opposite correlations with temperature in soil (positive) and marine (negative) environments. This phenomenon was probably driven by differences in nutrient availability, as GC content increases with nutrients while nutrients decrease with temperature in marine samples. By integrating these observations, we identified specific tetranucleotides, with 50% GC, that displayed consistent and robust temperature correlations across environments and may have contributed to the stability of predictions. This work highlights metagenome-wide DNA-temperature associations, relevant for understanding microbial community responses to global changes.

Journal Article

Cultivation of Ureaplasma urealyticum in continuous culture.

Continuous culture of Ureaplasma urealyticum is reported with a steady-state cell biomass of greater than 10(6) cells per ml. Thus, large cell numbers can be easily obtained; in addition, the system provides a powerful means for exploring what nutrients(s) limits the growth yield of this organism. Urea is shown not to be the growth-limiting nutrient in conventional media, although when provided in excess it appears to be completely hydrolyzed.

Bacteriological Techniques

Relationship between iron-limited growth and energy limitation during phased cultivation of Candida utilis.

The yeast Candida utilis was continuously synchronized by the phasing technique (6 h doubling time) with either iron or nitrogen as the limiting nutrient. Iron limitations resulted in decreased molar growth yields with respect to the carbon substrates and ammonia and in increased specific rates of oxygen uptake. Relatively low energy-charge values were maintained by the iron-limited culture. All these taken together seemed to indicate that the growth of the yeast under iron limitation was also limited by metabolically available energy. Consideralbe amounts of ethyl acetate were produced by the yeast under phased cultivation when the growth was limited by iron but not by nitrogen. In vitro studies using cell-free extracts showed that the substrates for ethyl acetate synthesis were acetyl coenzyme A (acetyl CoA) and ethanol. Under iron-limited growth acetyl CoA seemed to be diverted to ethyl acetate formation rather than being oxidized through the tricarboxylic acid (TCA) cycle. The possibility of energy limitation under iron-limited growth being brought about by the reduced capacity of the yeast to oxidize acetyl CoA through the TCA cycle is considered.

Acetates

Variations in the adenylate energy charge during phased growth (cell cycle) of Candida utilis under energy excess and energy-limiting growth conditions.

The variations in the levels of adenine nucleotides during the phased growth (cell cycle) of the yeast Candida utilis growing under nitrogen, sulfate, or iron limitation with glycerol as carbon source have been determined. Synchronous cultures were obtained by the continuous phasing technique, and the results were compared with those of chemostat cultures growing at similar growth rates and under the same types of nutrient limitation. Whereas the chemostat experiments indicated only the average energy status of cultures growing at random, results from phased cultures showed that the adenylate energy charge, defined as (ATP + (1/2)ADP)/(ATP + ADP + AMP) (where ATP, ADP, and AMP signify adenosine 5'-triphosphate, -diphosphate, and -monophosphate, respectively), varied during the phased growth of the yeast. These variations were related to the stage of development of the cells and to the type of nutrient limitation. In every case the energy charge dropped to a low value during the first half of the phasing cycle (cell cycle). Whereas the energy charge was maintained at relatively high levels (ranging from 0.78 to 0.94), for sulfate- or nitrogen-limited cultures, it was very low when iron was the growth-limiting nutrient (0.44 to 0.78). In spite of the low energy charge, the yeast continued to grow under iron limitation. The main component of the adenylate pool of the iron-limited culture was ADP and not ATP as observed with other types of nutrient limitation. It is concluded that under iron limitation the growth of the organism is limited by energy and that under energy-limited growth the energy charge of a growing organism is maintained at low levels. The reason for maintaining a low energy charge in an energy-limited culture is discussed.

Adenine Nucleotides

Morphological instability and roughening of growing 3D bacterial colonies.

How do growing bacterial colonies get their shapes? While colony morphogenesis is well studied in two dimensions, many bacteria grow as large colonies in three-dimensional (3D) environments, such as gels and tissues in the body or subsurface soils and sediments. Here, we describe the morphodynamics of large colonies of bacteria growing in three dimensions. Using experiments in transparent 3D granular hydrogel matrices, we show that dense colonies of four different species of bacteria generically become morphologically unstable and roughen as they consume nutrients and grow beyond a critical size-eventually adopting a characteristic branched, broccoli-like morphology independent of variations in the cell type and environmental conditions. This behavior reflects a key difference between two-dimensional (2D) and 3D colonies; while a 2D colony may access the nutrients needed for growth from the third dimension, a 3D colony inevitably becomes nutrient limited in its interior, driving a transition to unstable growth at its surface. We elucidate the onset of the instability using linear stability analysis and numerical simulations of a continuum model that treats the colony as an "active fluid" whose dynamics are driven by nutrient-dependent cellular growth. We find that when all dimensions of the colony substantially exceed the nutrient penetration length, nutrient-limited growth drives a 3D morphological instability that recapitulates essential features of the experimental observations. Our work thus provides a framework to predict and control the organization of growing colonies-as well as other forms of growing active matter, such as tumors and engineered living materials-in 3D environments.

Models, Biological

Streptococcus pneumoniae adaptation to nutrient deprivation and immune modulation drives upper respiratory tract colonization.

Streptococcus pneumoniae is a successful colonizer of the human upper respiratory tract; however, the mechanisms that enable its persistence in this nutrient-limited environment, with numerous immune mechanisms in place, remain enigmatic. Here, we examined how pneumococci adapt to upper respiratory tract conditions and how this affects host interactions. We measured intranasal metal ion and monosaccharide concentrations to create an in vivo-mimicking medium for studying pneumococcal adaptation. Growth in this medium was reduced compared to glucose-rich chemically defined media (CDM). Proteome analysis revealed a shift to galactose as the major carbohydrate source, and decreased levels of fatty acid biosynthesis proteins and pneumolysin, compared to other CDMs. Glycerophosphocholine accumulated extracellularly leading to decreased C-reactive protein and Immunoglobulin M binding to pneumococci. Pneumococci grown in in vivo-mimicking medium, compared to glucose-rich media, were more capable colonizers of primary epithelium and induced less epithelial cytokine release. Together, this shows how pneumococci adapt to the nutrient-limited respiratory environment, modulate epithelial cells, and evade humoral responses to facilitate persistent colonization.

Streptococcus pneumoniae

Copper and iron engage distinct metabolic programs for cellular survival.

Copper and iron are redox-active micronutrients with tightly coupled homeostasis, yet how copper modulates iron-dependent stress responses remains unclear. Using Saccharomyces cerevisiae under nutrient-limited conditions, we uncoupled proliferative growth from long-term survival to dissect metal-dependent adaptation. Copper selectively preserved survival without affecting growth, whereas iron showed similar effects. Iron chelation impaired growth and suppressed electron transport chain gene expression; copper partially rescued these defects but required iron availability for its pro-survival activity. Despite this interdependence, copper and iron engaged distinct signaling programs. Iron-dependent survival required a Target of Rapamycin complex 1 (TORC1)-permissive state and was attenuated by rapamycin, whereas copper remained active under TORC1 inhibition. In contrast, copper promoted survival through AMP-activated protein kinase (AMPK) and antioxidant pathways, while iron exhibited context-dependent AMPK reliance. Together, these findings reveal that copper and iron support cellular survival through distinct metabolic programs and suggest that the consequences of micronutrient availability are shaped by the underlying nutrient-sensing and metabolic state of the cell. This framework provides insight into how alterations in micronutrient homeostasis and metabolic signaling may influence cellular resilience during aging.

AMPK

Borrelia burgdorferi loses essential genetic elements and cell proliferative potential during stationary phase in culture but not in the tick vector.

The Lyme disease agent Borrelia burgdorferi is a polyploid bacterium with a segmented genome in which both the chromosome and over 20 distinct plasmids are present in multiple copies per cell. This pathogen can survive for at least 9 months in its tick vector in an apparent dormant state between blood meals, without losing cell proliferative capability when re-exposed to nutrients. Cultivated B. burgdorferi cells grown to stationary phase or resuspended in nutrient-limited media are often used to study the effects of nutrient deprivation. However, a thorough assessment of the spirochete's ability to recover from nutrient depletion has been lacking. Our study shows that starved B. burgdorferi cultures rapidly lose cell proliferative ability. Loss of genetic elements essential for cell proliferation contributes to the observed proliferative defect in stationary phase. The gradual decline in copies of genetic elements is not perfectly synchronized between chromosomes and plasmids, generating cells that harbor one or more copies of the essential chromosome but lack all copies of one or more non-essential plasmids. This phenomenon likely contributes to the well-documented issue of plasmid loss during in vitro cultivation of B. burgdorferi. In contrast, B. burgdorferi cells from ticks starved for 14 months showed no evidence of reduced cell proliferative ability or plasmid loss. Beyond their practical implications for studying B. burgdorferi, these findings suggest that the midgut of the tick vector offers a unique environment that supports the maintenance of B. burgdorferi's segmented genome and cell proliferative potential during periods of tick fasting.IMPORTANCEBorrelia burgdorferi causes Lyme disease, a prevalent tick-borne illness. B. burgdorferi must survive long periods (months to a year) of apparent dormancy in the midgut of the tick vector between blood meals. Resilience to starvation is a common trait among bacteria. However, this study reveals that, in laboratory cultures, B. burgdorferi poorly endures starvation and rapidly loses viability. This decline is linked to a gradual loss of genetic elements required for cell proliferation. These results suggest that the persistence of B. burgdorferi in nature is likely shaped more by unique environmental conditions in the midgut of the tick vector than by an innate ability of this bacterium to endure nutrient deprivation.

Borrelia burgdorferi

Dynamic Rhizodeposition in the Woody Perennial Populus trichocarpa.

Plants undergo physiological and metabolic changes that release specific molecules into the surrounding soil, a process collectively known as rhizodeposition. These compounds play crucial roles in plant-microbe-soil interactions, such as supporting plant development and resilience in changing environments. Under nutrient-limited conditions, these plant-derived compounds modify the rhizosphere environment, mobilizing otherwise inaccessible nutrients and recruiting stress-adaptive microbial communities that support stress resilience. Currently, the chemical diversity of rhizodeposition has yet to be fully realized but is expected to be a complex mixture that includes soluble organic compounds excreted from root cells, along with products of root cell turnover, sloughed-off root cap and border cells, and mucilage. Here, we developed a methodological and conceptual framework for an in-depth measurement of rhizodeposition through critical advancements in untargeted metabolomics. This approach provided foundational insights into the dynamic changes in rhizodeposition for the woody perennial Populus trichocarpa and rhizodeposit profiles varying by genotype, time, location, and environment. More broadly, this study provides a framework that will help formulate the next steps to effectively study rhizodeposition.

Populus

Genome-based predictions of metabolic preferences and substrate phenotypes in psychrotrophic bacteria from permafrost environments.

Genomes reveal vast functional potential, but harbor genomic noise that obscures prediction of metabolic and environmental preferences. Genomic databases are skewed towards clinically relevant and easily cultivated bacteria, limiting predictions for diverse and underrepresented environmental taxa. Psychrotrophic bacteria, which can survive and grow in cold, nutrient-limited, dry, and saline environments, are especially underrepresented despite their relevance for understanding microbial responses to changing cold environments and potential biotechnological value given growth at low temperatures. Assembling complete genomes of 48 isolates from Alaskan permafrost, seasonally frozen active layer soils, and terrestrial ice, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog annotations to evaluate the predictability of metabolic resource-use traits observed using phenotypic tests. Genome-predicted values for glycolytic versus gluconeogenic catabolic preference index, or sugar-acid preference (SAP), explained over 50% of the variance in empirically observed SAP. SAP was inversely correlated to genomic GC content, which follows phylum-level trends, indicating that coarse metabolic preference covaries with phylogeny. Regularized elastic net models offered a more granular view, linking KEGG genes to specific substrate utilization and sensitivity phenotypes and yielding moderate but reproducible accuracy (AUC 0.70-0.79) for 11 substrates, demonstrating that specific substrate responses may be predictable from relatively small subsets of KO genes. These results extend recent advances, such as the SAP metric, and highlight associations among genomic GC content, phylum, and broad metabolic strategy. Linking genomic content to phenotype using isolates is a necessary step toward predictive models of microbial function in environmental communities, and this work can be used for hypothesis generation, with applications towards more expansive data sets.IMPORTANCECold region soils and ice host psychrotrophic bacteria with metabolic traits and adaptations that enable persistence in harsh, resource-limited environments. However, these taxa are underrepresented in genomic reference databases dominated by well-studied, mesophilic organisms. This gap limits inference of ecological strategies and our ability to predict how these microbes may influence the large, thaw-vulnerable carbon reservoirs in permafrost. Here, we show that genomic GC content is associated with the sugar-versus-acid catabolic preference (SAP) of isolates across major phyla, suggesting that broad genomic features may provide a coarse signal of metabolic strategy. We demonstrate that a modified SAP metric, using binary (positive/negative) substrate utilization rather than detailed growth rate measurements, is moderately predictive, thus extending its application to slow-growing or difficult-to-culture taxa. Together, these advances broaden the toolkit for linking genome content to resource-use traits (phenotype) in poorly characterized, cold-adapted bacteria and offer a tractable entry point to broad prediction and hypothesis generation.

Genome, Bacterial

TCF25 serves as a nutrient sensor to orchestrate metabolic adaptation and cell death by enhancing lysosomal acidification under glucose starvation.

Cells adapt to nutrient limitation by activating catabolic and inhibiting anabolic pathways, yet prolonged stress may lead to cell death. How cells orchestrate metabolic adaptation and cell death to nutrient stress is poorly understood. We conduct a genome-wide CRISPR-Cas9 screen to identify regulators in glucose-starvation-induced cell death and find a group of genes in lysosomal pathway is enriched following glucose starvation. We focus on one candidate gene, Transcriptional Factor 25 (TCF25). We find TCF25 enhances lysosomal acidification by targeting V-ATPase, promoting autophagy and ATP generation under glucose starvation. However, prolonged glucose starvation constitutively activates ferritinophagy via TCF25, increasing lysosomal membrane permeability (LMP) and leading to lysosome-dependent cell death (LDCD). Knocking out TCF25 or V-ATPase components prevents cell death. Furthermore, TCF25 deficiency protects mice from hepatic ischemia-reperfusion injury. Our findings identify TCF25 as a crucial nutrient sensor that regulates lysosomal activity, offering potential therapeutic targets for metabolic and ischemic disorders.

Lysosomes

Control of lactate production by Selenomonas ruminantium: homotropic activation of lactate dehydrogenase by pyruvate.

Selenomonas ruminantium produced one mole of D(-)-lactate per mole of glucose used at all dilution rates in ammonia-limited continuous culture. In contrast, lactate production varied according to the dilution rate when glucose was the limiting nutrient. At dilution rates of less than 0.2 h-1, acetate and propionate were the main fermentation products and lactate production was low. At dilution rates above 0.2 h-1, the pattern changed to one of high lactate production similar to that under ammonia limitation. Experiments with cell-free extracts of S. ruminantium showed that D(-)-lactate dehydrogenase had sigmoidal kinetics consistent with homotropic activation of the enzyme by its substrate, pyruvate. This feature allows S. ruminantium to amplify the effects of relatively small changes in the intracellular concentration of pyruvate to cause much larger changes in the rate of production of lactate. Some confirmation that this mechanism of control occurs under physiological conditions was obtained in glucose-limited culture, in which the sigmoidal increase in lactate production was accompanied by a linear increase in pyruvate excretion as the dilution rate increased.

Bacteria

Enzyme kinetics shapes the growth response of metabolic networks.

Microbes adjust their metabolism to environmental challenges by changing protein expression levels, metabolite concentrations, and reaction rates. Average expression levels in large proteome sectors change coherently, while individual proteins show divergent shifts even within the same pathway. Here, we establish a metabolic model that integrates local enzyme kinetics and global network architecture to predict the joint growth response of proteins and metabolites. Under nutrient limitation, we predict a remarkably simple pattern of proteome reallocation with growth rate: protein expression levels change linearly but heterogeneously. For a given enzyme, the direction of change is determined by its local kinetic constants - catalytic rate and substrate affinity - and by the degree of nutrient restriction affecting its embedding pathway. This double-graded growth response of the proteome is mediated by restriction-dependent metabolite levels, which are predicted to decrease with growth rate in a nonlinear way. The model establishes three specific growth laws: protein expression changes of individual enzymes are negatively correlated with their expression and with their substrate saturation at high growth; average changes of pathways and larger functional sectors are correlated with their internal variance. These predictions are in quantitative agreement with measured system-wide proteomics and metabolomics data of E. coli. Enzyme-specific response patterns are a starting point for model-guided interventions into bacterial metabolism.

Kinetics