Late Stationary Phase Research Articles

Identification and characterization of a novel lytic peptidoglycan transglycosylase (MltC) in Shigella dysenteriae.

Shigellosis remains a worldwide health problem due to the lack of vaccines and the emergence of antibiotic-resistant strains. Shigella (S.) dysenteriae has rigid peptidoglycan (PG), and its tight regulation of biosynthesis and remodeling is essential for bacterial integrity. Lytic transglycosylases are highly conserved PG autolysins in bacteria that play essential roles in bacterial growth. However, their precise functions are obscure. We aimed to identify, clone, and express MltC, a unique autolysin in Escherichia (E.) coli C41 strain. The purification of recombinant MltC protein was performed using affinity chromatography and size-exclusion chromatography methods. The PG enzymatic activity of MltC was investigated using Zymogram and Fluorescein isothiocyanate (FITC)-labeled PG assays. Also, we aimed to detect its localization in bacterial fractions (cytoplasm and membrane) by western blot using specific polyclonal anti-MltC antibodies and its probable partners using immunoprecipitation and mass spectrometry applications. Purified MltC showed autolysin activity. Native MltC showed various locations in S. dysenteriae cells during different growth phases. In the Lag and early stationary phases, MltC was not found in cytoplasm and membrane fractions. However, it was detected in cytoplasm and membrane fractions during the exponential phase. In the late stationary phase, MltC was expressed in the membrane fraction only. Different candidate protein partners of MltC were identified that could be essential for bacterial growth and pathogenicity. This is the first study to suggest that MltC is indeed autolysin and could be a new drug target for the treatment of shigellosis by understanding its biological functions.

CCCP Facilitates Aminoglycoside to Kill Late Stationary-Phase Escherichia coli by Elevating Hydroxyl Radical.

Improving the efficacy of existing antibiotics is significant for combatting antibiotic resistance that poses a major threat to human health. Carbonyl cyanide m-chlorophenylhydrazine (CCCP), a well-known protonophore for dissipating proton motive force (PMF), has been widely used to block the PMF-dependent uptake of aminoglycoside antibiotics and thus suppress aminoglycoside lethality. Here, we report that CCCP and its functional analog FCCP, but not other types of protonophores, unprecedently potentiate aminoglycosides (e.g., tobramycin and gentamicin) by 3-4 orders of magnitude killing of Escherichia coli, Staphylococcus aureus, Shigella flexneri, and Vibrio alginolyticus cells in stationary phase but not these cells in exponential phase nor other 12 bacterial species we examined. Overall, the effect of CCCP on aminoglycoside lethality undergoes a gradual transition from suppression against E. coli exponential-phase cells to potentiation against late stationary-phase cells, with the cell growth status and culture medium being crucial. Consistently, disturbance of the PMF by changing transmembrane proton gradient (ΔpH) or electric potential (ΔΨ) also potentiates tobramycin. Nevertheless, CCCP neither increases the intracellular concentration of tobramycin nor decreases the MIC of the antibiotic, thus excluding that CCCP acts as an efflux pump inhibitor to potentiate aminoglycosides. Rather, we show that the combined treatment dramatically enhances the cellular level of hydroxyl radical under both aerobic and anaerobic culturing conditions, under which the antioxidant N-acetyl cysteine fully suppresses both hydroxyl radical accumulation and cell death. Together, these findings open a new avenue to develop certain protonophores as aminoglycoside adjuvants against pathogens in stationary phase and also illustrate an essential role of hydroxyl radical in aminoglycoside lethality regardless of aerobic respiration.

Chronological age-related metabolome responses in the dinoflagellate Karenia mikimotoi, can predict future bloom demise

Karenia mikimotoi is a common harmful algal bloom (HAB)-forming dinoflagellate and has caused severe financial loss in aquaculture. There are limited metabolomic studies on dinoflagellate biology. Here, we examined alterations in metabolic profiles over the growth curve of K. mikimotoi under nitrogen or phosphorus deficiency and further explored a key criterion for the diagnosis of late stationary phase to identify when the dinoflagellate cells will enter bloom demise. The results demonstrate the differential expression of metabolites for coping with chronological aging or nutrient deprivation. Furthermore, an increase in the glucose to glycine ratio in the late stationary phase was indicative of dinoflagellate cells entering bloom demise; this was also detected in the cultured diatom, Chaetoceros tenuissimus, indicating that this may be the general criterion for phytoplankton species. Our findings provide insights regarding chronological aging and the criterion for the prediction of phytoplankton bloom demise.

Growth stage-related biomass, pigments, and biochemical composition of Stichococcus bacillaris, Synechococcus sp., and Trentepohlia aurea isolated from Gua Tempurung, a cave in Malaysia

As part of the lampenflora that inhabit limestone caves, microalgae play an important role in cave ecosystems but are understudied in tropical ecoregions. In the present study, the dominant eukaryotic and prokaryotic microalgae identified in lampenflora samples collected from Gua Tempurung, a cave in Malaysia, and growth stage-related microalgal attributes were determined. Stichococcus bacillaris, Synechococcus sp., and Trentepohlia aurea were selected and cultured in Bold's Basal Medium (S. bacillaris and T. aurea) or BG-11 medium (Synechococcus sp.) under laboratory conditions. The highest specific growth rate (0.72 ± 0.21 day−1) and dry weight (0.11 ± 0.04 mg L−1) were recorded in S. bacillaris in the early stationary phase. Trentepohlia aurea and Synechococcus sp. had the highest ash-free dry weight and total ash percentage (11.18 ± 4.64 mg L−1 and 8.55% ± 6.73%, respectively) in the early stationary phase. Stichococcus bacillaris had the highest moisture content (84.26% ± 0.64%) in the exponential phase. Chlorophylls a and b were highest in the early stationary phase in T. aurea (0.706 ± 0.40 mg L−1 and 1.094 ± 0.589 mg L−1, respectively). Carotenoid levels were highest in Synechococcus sp. in the early stationary stage (0.07 ± 0.02 mg L−1). Lipids were the major biochemical compound identified at the highest levels in Synechococcus sp. (67.87% ± 7.75%) in the early stationary phase, followed by protein recorded at the highest levels in T. aurea (57.99% ± 4.99%) in the early stationary phase. Carbohydrates were the compound identified least often with the highest recorded levels found in T. aurea (9.94% ± 0.49%) in the late stationary phase. Biomass, pigments, and biochemical accumulation varied at different growth stages in the studied microalgae, and this variation was species-specific. The present study provides a benchmark for the growth phases of aerophytic cave microalgae, which will be useful for determining their optimum harvest time and obtaining biochemical compounds of interest.

Assessment of the recovery and photosynthetic efficiency of Breviolum psygmophilum and Effrenium voratum (Symbiodiniaceae) following cryopreservation.

Many strains of Symbiodiniaceae have been isolated and their genetics, taxonomy, and metabolite production studied. Maintaining these cultures requires careful and regular sub-culturing that is costly with a high risk of species contamination or loss. Cryopreservation is a viable alternative for their long-term storage; however, there is uncertainty as to whether cryopreservation impacts the photosynthetic performance of Symbiodiniaceae. We investigated the growth rates and photosynthetic efficiency of two species, Breviolum psygmophilum and Effrenium voratum before and after cryopreservation. Rapid light curves (RLCs) produced using Pulse Amplitude Modulated (PAM) fluorometry were used to generate detailed information on the characteristics of photosystem II (PSII). The maximum electron transport rate (ETRmax) and the quantum yield (Fv/Fm) of the control (non-cryopreserved) and cryopreserved culture isolates were assessed across the growth cycle. The non-cryopreserved isolate of B. psygmophilum had a higher quantum yield than the cryopreserved isolate from day 12 to day 24, whereas there were no differences from day 28 to the late stationary phase. There were no significant differences in ETRmax. No significant differences were observed in quantum yield or ETRmax between the control and cryopreserved E. voratum isolates. The ability of cryopreserved strains to recover and regain their photosynthetic efficiency after freezing demonstrates the utility of this method for the long-term storage of these and other Symbiodiniaceae species.

Oxidation of biological molecules with age and induced oxidative stress in different growth phases of Saccharomyces cerevisiae.

One of the mechanistic approaches for explaining ageing is the oxidative stress theory of ageing. Saccharomyces cerevisiae has been used as a model to study ageing due to many factors. We have attempted to investigate if the differential ability to withstand oxidative stress can be correlated with their lifespans. In all the four strains studied (AP22, 699, 8C, and SP4), there was no age-associated increases in lipid peroxidation levels measured as thiobarbituric acid reactive substances (TBARS). Under induced oxidative stress conditions, there was an increased TBARS level in both the ages assessed with a quantum-fold increase in the stationary phase cells of AP22.In contrast, the late stationary phase cells of 8C exhibited the least susceptibility to induced oxidative stress. The level of TBARS in both exponential and late stationary phase cells of 699 was overall more than that in the other three strains. Protein carbonylation increased with age in 8C and 699. Induced stress increased carbonylation in the exponential cells of SP4 and 699 and the stationary phase cells of all four strains. Protein carbonylation data indicate that the AP22 cells exhibit decreased protein carbonylation vis-à-vis the other strains. Induced stress data showed that while the exponential cells of 699 are susceptible, the late stationary phase cells of 699 are most resistant. Western blotting analysis using anti-HNE antibodies showed two proteins of molecular mass ~ 56 and ~ 84kDa that were selectively modified with age in all the strains. Under induced stress conditions, an additional protein of ~ 69kDa was oxidized. Our investigation shows that the difference in lifespan between the four strains of S. cerevisiae may be regulated by oxidative stress. Knowledge of the identity of the oxidized proteins will significantly facilitate a better understanding of the effect of oxidative stress conditions on the cells of S. cerevisiae.

Abiotic and biotic factors controlling sexual reproduction in populations of Pseudo-nitzschia pungens (Bacillariophyceae)

Pseudo-nitzschia pungens is a widely distributed marine pennate diatom. Hybrid zones, regions in which two different genotypes may interbreed, are important areas for speciation and ecology, and have been reported across the globe for this species. However, sexual reproduction between differing clades in the natural environment is yet to be observed and is difficult to predict. Here we carried out experiments using two mono-clonal cultures of P. pungens from different genotypes to measure the frequency and timing of sexual reproduction across varying biotic (growth phases and cell activity potential) and abiotic conditions (nutrients, light, turbulence). We found the mating rates and number of zygotes gradually decreased from exponential to late stationary growth phases. The maximum zygote abundance observed was 1,390 cells mL−1 and the maximum mating rate was 7.1%, both which occurred during the exponential growth phase. Conversely, only 9 cells mL−1 and a maximum mating rate of 0.1% was observed during the late stationary phase. We also found the higher the relative potential cell activity (rPCA) in parent cells, as determined by the concentration of chlorophyll a per cell and the ratio of colony formation during parent cultivations, revealed higher mating rates. Furthermore, sexual events were reduced under nutrient enrichment conditions, and mating pairs and zygotes were not formed under aphotic (dark) or shaking culture conditions (150 rpm). In order to understand the sexual reproduction of Pseudo-nitzschia in the natural environment, our results highlight that it is most likely the combination of both biotic (growth phase, Chl. a content) and abiotic factors (nutrients, light, turbulence) that will determine the successful union of intraspecific populations of P. pungens in any given region.

Function of the mdxR gene encoding a novel regulator for carbohydrate metabolism and sporulation in Bacillus subtilis 168.

The mdxR gene located upstream of mdxD, encoding a maltogenic amylase, has been annotated as a member of LacI-type transcriptional regulator in Bacillus subtilis 168 but its function has not been investigated yet. In this study, expression pattern of the mdxR promoter (PmdxR) and effects of mdxR were investigated to elucidate the function of mdxR. Expression of PmdxR was monitored by the β-galactosidase activity expressed from the PmdxR-lacZ fusion integrated at the amyE locus on the chromosome. The promoter was induced by starch, β-cyclomaltodextrin, or maltose at early exponential phase and kept expressed until late stationary phase. However, it was repressed by glucose, sucrose, or glycerol, suggesting that it was under catabolite repression. Furthermore, interactions of MdxR and Spo0A to the DNA fragment carrying PmdxR or PmdxD were detected by mobility-shift assay, implying that MdxR was a novel transcription regulator for both genes, which were regulated also by Spo0A. The mdxR mutant impaired the expressions of mdxD and malL (encoding an α-glucosidase); degraded accumulated glycogen slower than the wild type and the mdxD mutant. Both of the mdxR and the mdxD mutants formed more endospores (50.95% and 47.10%) than the wild type (23.90%). Enhanced sporulation by these mutations could be of industrial interest where sporulation or endospores of B. subtilis matters. These results indicate that MdxR functions as a transcriptional regulator for mdxR, mdxD, and other genes in the gene cluster that is related to the maltose/maltodextrin metabolism. MdxR and MdxD are also involved in glycogen metabolism and sporulation, tentatively by modulating the net energy balance in the cell.

Revealing the accessibility and surface morphology of the Escherichia coli nucleoid by genetically encoded live-cell single-particle tracking.

Membraneless organelles play a key role in the subcellular organization and regulation of bacterial cells. In particular, the bacterial genome is highly compacted into a membraneless organelle: the nucleoid. The structure and accessibility of the nucleoid significantly influences gene expression, and this morphology varies with growth phase and under various environmental conditions. Therefore, revealing how the DNA meshwork changes as a function of the growth phase will deepen our understanding of how bacteria regulate transcription in response to stress. To realize this goal, we applied single-particle tracking in E. coli and super-resolved the dynamics of an extrinsic 25-nm protein assembly with a well-defined dodecahedron structure: GFP-nanocages (Hsia et al., Nature 2016). By super-resolving the GFP-nanocage positions from log phase to late stationary phase, we observed an increasingly obvious exclusion of the probe by nucleoid, which indicates that the chromosome is compacting and becoming less accessible for gene expression. Our preliminary experiments also found a novel observation: the nucleoid size and compaction are not necessarily correlated. We hypothesize that at the intermediate stage of compaction, cells with small nucleoids may have a relatively low global compaction level because DNA chains are dragged by the force generated by DNA-NAP complex formation, leading to compacted rigid cores surrounded by a loosened DNA meshwork. By measuring the single-molecule step sizes at the periphery of the nucleoid, we understand the rigidity and smoothness of nucleoid surface at various phases. Furthermore, by analyzing the angle distribution within single-particle trajectories, we quantify the orientation of interactions with the nucleoid. The gene expression levels differ with cell growth stage (Verma et al., PLoS Genetics 2019). Our research provides a novel way of understanding how subcellular structure and gene expression are related.

Phenotypical Differences between Leishmania (Leishmania) amazonensis PH8 and LV79 Strains May Impact Survival in Mammal Host and in Phlebotomine Sand Flies.

We previously showed that L. (Leishmania) amazonensis promastigotes and amastigotes of the PH8 strain generated larger lesions in mice than LV79, and that lesion-derived amastigotes from the two strains differ in their proteomes. We recently reported that PH8 promastigotes are more phagocytized by macrophages. Promastigotes' membrane-enriched proteomes showed several differences, and samples of each strain clustered based on proteomes. In this paper, we show phenotypic differences between PH8 and LV79 promastigotes that may explain the higher virulence of PH8. We compared in vitro macrophage infections by day 4 (early) and day 6 (late stationary phase) cultures, resistance to complement, and LPG characteristics. PH8 promastigotes showed a higher infectivity and were more resistant to murine complement. LPG was different between the strains, which may influence the interaction with macrophages and survival to complement. We compared the infection of the permissive vector Lutzomyia longipalpis. PH8 was more abundant in the vector's gut 72 h after feeding, which is a moment where blood digestion is finished and the parasites are exposed to the gut environment. Our results indicate that PH8 promastigotes are more infective, more resistant to complement, and infect the permissive vector more efficiently. These data suggest that PH8 is probably better adapted to the sand fly and more prone to survive in the vertebrate host.

Growth Phase Influence the Gene Expression and Metabolite Production Related to Indole-3-Acetic Acid (IAA) Biosynthesis by Serratia plymuthica UBCF_13.

<b>Background and Objective:</b> The optimization of the indole-3-acetic acid (IAA) producing capability of <i>Serratia plymuthica</i> UBCF_13 has been intensively studied. This work tried to reveal the effect of growth phases on IAA production, gene expression and metabolite synthesis related to the IAA biosynthesis pathway. <b>Materials and Methods:</b> The growth curve and IAA production were measured every 3 hrs. The putative IAA biosynthesis pathway was investigated based on the UBCF_13 genome. To identify the possible pathway of IAA biosynthesis in UBCF_13, we applied the Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and High-Performance Liquid Chromatography (HPLC) analysis to measure the transcript levels of each gene and indole metabolite production based on tryptophan treatment at different times of incubation. <b>Results:</b> The optimal IAA production on colorimetric assay was at 9 hrs of incubation (initial stationary phase). The level expression of <i>puuC</i>, <i>DDC</i>, <i>oxdA</i>, <i>amiE</i>, <i>nthA</i> and <i>nthB</i> have been upregulated maximum in 3 hrs of culture time (lag phase), except <i>tyrB</i> and <i>ipdC</i>. The highest transcript level of the genes was found in nitrile hydratase genes (<i>nthA</i> and <i>nthB</i>) and indole-3- acetamide (IAM) has been detected as the only intermediate in the crude extract of UBCF_13 thus the IAM pathway may be used to produce IAA. The maximum IAA production on HPLC analysis was found at 21 hrs of incubation (late stationary phase). <b>Conclusion:</b> This study gives a new insight that the best time to measure gene expression and intermediates related to the IAA biosynthetic pathway in bacteria was found at a specific growth phase.

Growth and Physiological Responses of Chrysotila roscoffensis (Coccolithales) to Different Phosphorus Resources

The growth and physiological process of microalgae interact with environmental nutrients. In the present study, we compared the growth and physiological characteristics of coccolithophore Chrysotila roscoffensis under the effects of phosphate at high and low concentrations, and of organic phosphorus of adenosine triphosphate (ATP) and Na2-glycerophosphate (SG). The growth, photosynthesis, calcification, alkaline phosphatase activity, and inorganic nutrient absorption rate were comparatively studied. The results showed that the culture with a low concentration of initial phosphate (6 μM) had a lower growth rate than that with a high concentration (45 μM). However, a relatively similar maximum cell density could be achieved. Equimolar inorganic phosphorus supported a higher initial growth rate than organic phosphorus. ATP was better than SG at supporting higher growth rates, higher photosynthetic activity, and higher cell density. Cellular alkaline phosphatase (AP) responded rapidly to nutrient variations with sharp changes of activities, independent of the initial P resources. Cellular calcification was at a higher level in groups with lower growth rates. Phosphate in low concentration in the medium was not absorbed during the early growth period while that in high concentration was rapidly absorbed. Instead, phosphate in low concentration was rapidly absorbed in the late stationary phase. The absorption of nitrate was affected by the initial P resources as well. The DIN/DIP ratio in the water varied significantly during the growth periods. The results indicated that C. roscoffensis had flexible physiological strategies in utilizing varied phosphorus resources, and high cell density maintenance of C. roscoffensis may play roles in nutrient conditions in the water. This study may help to extend the understanding of nutrient utilization strategy in microalgae and to apply reference in the application of Chrysotila species in the removal of nutrient pollution.

Pseudomonas veronii strain 7–41 degrading medium-chain n-alkanes and polycyclic aromatic hydrocarbons

Pollution of the environment by crude oil and oil products (represented by various types of compounds, mainly aliphatic, mono- and polyaromatic hydrocarbons) poses a global problem. The strain Pseudomonas veronii 7–41 can grow on medium-chain n-alkanes (C8–C12) and polycyclic aromatic hydrocarbons such as naphthalene. We performed a genetic analysis and physiological/biochemical characterization of strain 7–41 cultivated in a mineral medium with decane, naphthalene or a mixture of the hydrocarbons. The genes responsible for the degradation of alkanes and PAHs are on the IncP-7 conjugative plasmid and are organized into the alk and nah operons typical of pseudomonads. A natural plasmid carrying functional operons for the degradation of two different classes of hydrocarbons was first described. In monosubstrate systems, 28.4% and 68.8% of decane and naphthalene, respectively, were biodegraded by the late stationary growth phase. In a bisubstrate system, these parameters were 25.4% and 20.8% by the end of the exponential growth phase. Then the biodegradation stopped, and the bacterial culture started dying due to the accumulation of salicylate (naphthalene-degradation metabolite), which is toxic in high concentrations. The activity of the salicylate oxidation enzymes was below the detection limit. These results indicate that the presence of decane and a high concentration of salicylate lead to impairment of hydrocarbon degradation by the strain.

Thallusin Quantification in Marine Bacteria and Algae Cultures.

Thallusin, a highly biologically active, phytohormone-like and bacterial compound-inducing morphogenesis of the green tide-forming macroalga Ulva (Chlorophyta), was determined in bacteria and algae cultures. A sensitive and selective method was developed for quantification based on ultra-high-performance liquid chromatography coupled with electrospray ionization and a high-resolution mass spectrometer. Upon C18 solid phase extraction of the water samples, thallusin was derivatized with iodomethane to inhibit the formation of Fe−thallusin complexes interfering with the chromatographic separation. The concentration of thallusin was quantified during the relevant phases of the bacterial growth of Maribacter spp., ranging from 0.16 ± 0.01 amol cell−1 (at the peak of the exponential growth phase) to 0.86 ± 0.13 amol cell−1 (late stationary phase), indicating its accumulation in the growth medium. Finally, we directly determined the concentration of thallusin in algal culture to validate our approach for monitoring applications. Detection and quantification limits of 2.5 and 7.4 pmol L−1, respectively, were reached, which allow for quantifying ecologically relevant thallusin concentrations. Our approach will enable the surveying of thallusin in culture and in nature and will thus contribute to the chemical monitoring of aquaculture.

Depiction of growth specific changes in concentration of storage products in centric marine diatom Chaetoceros gracilis

Chaetoceros, with >232 identified species is one of the largest genera of planktonic diatoms playing a pivotal role in aquatic ecosystem. Known for its high metabolite content and suitability as fish feed, C. gracilis, has been chosen for this study to comprehend the variation in carbon utilization and accumulation of reserve compounds at different growth stages of the alga. The uptake and influence of two discrete sources of silica on cellular growth and metabolite production was also analyzed. The result showed that the growth rate (0.27 d−1) and cell number (132 × 105 cells mL−1) of the cultures supplemented with nano-fabricated silica was higher than those with meta silicate. Concurrently both biomass and lipid productivity (0.082 and 0.043 g L−1 d−1) was also found to be higher in the former culture setup. The study further indicates both the extracellular polymeric substances (EPS), and storage lipid content was maximized during the late exponential and stationary phase. Though the bound EPS content was higher in the cultures supplemented with nano silica (0.095 g L−1), the level of unbound EPS and lipid % dry weight was higher in cultures with sodium meta silicate (1.33 g L−1 and 40.25%, respectively). Thus, the results provide evidence that how co-accumulation of metabolites are closely correlated to the growth cycle of diatoms and that commercial production of such highly valuable compounds can be achieved by targeting the specific growth phase even in outdoor setups.

Impact of Environmental Conditions on the Protein Content of Staphylococcus aureus and Its Derived Extracellular Vesicles.

Staphylococcus aureus, a major opportunistic pathogen in humans, produces extracellular vesicles (EVs) that are involved in cellular communication, the delivery of virulence factors, and modulation of the host immune system response. However, to date, the impact of culture conditions on the physicochemical and functional properties of S. aureus EVs is still largely unexplored. Here, we use a proteomic approach to provide a complete protein characterization of S. aureus HG003, a NCTC8325 derivative strain and its derived EVs under four growth conditions: early- and late-stationary growth phases, and in the absence and presence of a sub-inhibitory concentration of vancomycin. The HG003 EV protein composition in terms of subcellular localization, COG and KEGG categories, as well as their relative abundance are modulated by the environment and differs from that of whole-cell (WC). Moreover, the environmental conditions that were tested had a more pronounced impact on the EV protein composition when compared to the WC, supporting the existence of mechanisms for the selective packing of EV cargo. This study provides the first general picture of the impact of different growth conditions in the proteome of S. aureus EVs and its producing-cells and paves the way for future studies to understand better S. aureus EV production, composition, and roles.

Effect of high CO2 concentrations on Desmodesmus abundans RSM lipidome

Lipids are of interest as potential byproducts of CO2 mitigation systems. We characterize the effect of different CO2 concentrations in air (0.04 % pure air, 25 % and 50 % v/v CO2/air) on Desmodesmus abundans RSM lipidome, adapted for eight years to these conditions. Batch cultures grew similarly but more biomass was generated at high CO2 (1.2- to 1.9-fold increase). Lipidome analysis at late exponential, and early and late stationary phase revealed 100 putative lipid features with significant changes grouped into five distinct clusters. Clusters 1 and 2 characterized by glycerolipids (GL, 66–100 %) and clusters from 3 to 5 as glycerophospholipids (GP, 76–100 %). Under air (0.04 % CO2), most features of clusters 1, 2, and 5 were nearly constant through time, but under high CO2, lipid profiles seemed to be determined by dCO2 availability. At dCO2 depletion, GL probably acts as a precursor of GP. During stationary phase, distinct cell maintenance strategies seemed to occur between cultures as lipids or starch were consumed at 25 % and 50 % CO2 to generate GP or GL, respectively. Also, under high CO2, both cultures exhibited an increase in pigments but only under 25 % CO2 a 1.6-fold increase in lipids was observed, which were later consumed. Results suggest different responses to CO2 among cultures; however, at this moment, it is not possible to elucidate whether differences are caused by CO2 treatment or long-term acclimation, or both. Studies should evaluate selected cultures under particular growth conditions to generate specific lipid byproducts for application.

Changes to the mtDNA copy number during yeast culture growth.

We show that the mitochondrial DNA (mtDNA) copy number in growing cultures of the yeast Saccharomyces cerevisiae increases by a factor of up to 4, being lowest (approx. 10 per haploid genome) and stable during rapid fermentative growth, and highest at the end of the respiratory phase. When yeast are grown on glucose, the onset of the mtDNA copy number increase coincides with the early stages of the diauxic shift, and the increase continues through respiration. A lesser yet still substantial copy number increase occurs when yeast are grown on a nonfermentable carbon source, i.e. when there is no diauxic shift. The mtDNA copy number increase during and for some time after the diauxic shift is not driven by an increase in cell size. The copy number increase occurs in both haploid and diploid strains but is markedly attenuated in a diploid wild isolate that is a ready sporulator. Strain-to-strain differences in mtDNA copy number are least apparent in fermentation and most apparent in late respiration or stationary phase. While changes in mitochondrial morphology and function were previously known to accompany changes in physiological state, it had not been previously shown that the mtDNA copy number changes substantially over time in a clonal growing culture. The mtDNA copy number in yeast is therefore a highly dynamic phenotype.

Proteomic profiling of the carbon-starved Escherichia coli reveals upregulation of stress–inducible pathways implicated in biological adhesion and methylglyoxal metabolism

Starvation in bacteria is a complex adaptive response to deprivation of nutrients that has been shown to implicate a number of stress networks that modulate pathogenicity and antibiotic resistance. Starvation in nature is qualitatively different from in-culture late stationary phase energy source depletion. To look into proteome-level alterations elicited by complete elimination of carbon source, we used Escherichia coli HT115-derived SLE1 strain cells and a combination of label-free and metabolic isotope labeling approaches. We isolated pathways differentially affected by carbon starvation and observed robust upregulation of proteins implicated in networks belonging to Gene Ontology terms ‘Biological adhesion’ and ‘Methylglyoxal metabolic process’.

Biogenesis of selenium nanospheres using Halomonas venusta strain GUSDM4 exhibiting potent environmental applications.

Selenite reducing bacterial strain (GUSDM4) isolated from Mandovi estuary of Goa, India was identified as Halomonas venusta based on 16S rRNA gene sequence analysis. Its maximum tolerance level for sodium selenite (Na2SeO3) was 100mM. The 2, 3-diaminonaphthalene-based spectroscopic analysis demonstrated 96 and 93% reduction of 2 and 4mM Na2SeO3 respectively to elemental selenium (Se0) during the late stationary growth phase. Biosynthesis of Se nanoparticles (SeNPs) commenced within 4h during the log phase, which was evident from the brick red color in the growth medium and a characteristic peak at 265nm revealed by UV-Vis spectrophotometry. The intracellular periplasmic synthesis of SeNPs in GUSDM4 was confirmed by transmission electron microscopy (TEM). Characterization of SeNPs by X-ray crystallography, TEM and energy-dispersive X-ray analysis(EDAX) clearly demonstrated spherical SeNPs of 20-80nm diameter with hexagonal crystal lattice. SeNPs (0.8 and 1mg/L) primed seeds under arsenate [As(V)] stress showed increase in shoot length, root length and biomass by 1.4-, 1.5- and 1.1-fold respectively, as compared to As(V) primed seeds alone. The proline and phenolic content in seeds primed with SeNPs under arsenate stress showed alleviated levels proving its ameliorative potential. SeNPs also demonstrated anti-biofilm activity at 20µg/mL against human pathogens which was evident by scanning electron microscopic (SEM) analysis. SeNPs interestingly revealed mosquito larvicidal activity also. Therefore, these studies have clearly demonstrated amazing potential of the marine bacterium, Halomonas venusta in biosynthesis of SeNPs and their applications as ameliorative, anti-biofilm and mosquito larvicidal agents which is the first report of its kind.