Stationary Growth Phase Research Articles

How do microalgae in response to biological pollution treat in cultivation? A case study investigating microalgal defense against ciliate predator Euplotes vannus.

Microalgae have significant amounts of proteins, lipids, carotenoids, vitamins, minerals, and unique pigments. However, with the gradual expansion of microalgae cultivation, hostile biological pollution seriously restricted the large-scale microalgae cultivation and limited the exploitation of its biological resources. Moreover, protozoan poses the greatest threat to microalgae cultivation. Here, the relationship between six marine economic microalgae populations and their ciliate predator Euplotes vannus was examined. And four concentrations were designed for each type of microalgae to carry out the experiment. It was revealed that four species of microalgae inhibit the ciliate population growth at high density. Furthermore, the experiment which was the influence of microalgae at three different growth stages on the growth of the ciliates for these four kinds of high-density inhibitory microalgae was designed. The microalgae inhibitory effects were already exhibited at the end of the exponential growth phase, and it was significantly inhibited during the stationary growth phase. As the microalgae concentration increased, the inhibitory effect became more pronounced. This study provides fundamental data for screening protozoan-inhibiting microalgae and shows potential to be used in algae cultivation.

Clostridioides difficile Phosphoproteomics Shows an Expansion of Phosphorylated Proteins in Stationary Growth Phase.

ABSTRACTPhosphorylation is a posttranslational modification that can affect both housekeeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile, the extent and nature of phosphorylation events are poorly characterized, though a protein kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine, and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of the stationary growth phase. Our approach identifies expected targets and phosphorylation sites among the more than 1,500 phosphosites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV), and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis.IMPORTANCE In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.

Insight Into the Anti-staphylococcal Activity of JBC 1847 at Sub-Inhibitory Concentration.

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus, using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847–treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound.

A peculiar cell cycle arrest at g2/m stage during the stationary phase of growth in the wine yeast Hanseniaspora vineae.

Yeasts of the genus Hanseniaspora gained notoriety in the last years due to their contribution to wine quality, and their loss of several genes, mainly related to DNA repair and cell cycle processes. Based on genomic data from many members of this genus, they have been classified in two well defined clades: the "faster-evolving linage" (FEL) and the "slower-evolving lineage" (SEL). In this context, we had detected that H. vineae exhibited a rapid loss of cell viability in some conditions during the stationary phase compared to H. uvarum and S. cerevisiae. The present work aimed to evaluate the viability and cell cycle progression of representatives of Hanseniaspora species along their growth in an aerobic and discontinuous system. Cell growth, viability and DNA content were determined by turbidity, Trypan Blue staining, and flow cytometry, respectively. Results showed that H. uvarum and H. opuntiae (representing FEL group), and H. osmophila (SEL group) exhibited a typical G1/G0 (1C DNA) arrest during the stationary phase, as S. cerevisiae. Conversely, the three strains studied here of H. vineae (SEL group) arrested at G2/M stages of cell cycle (2C DNA), and lost viability rapidly when enter the stationary phase. These results showed that H. vineae have a unique cell cycle behavior that will contribute as a new eukaryotic model for future studies of genetic determinants of yeast cell cycle control and progression.

In vivo quantification of polyhydroxybutyrate (PHB) in the alphaproteobacterial methanotroph, Methylocystis sp. Rockwell

Methane is a common industrial by-product that can be used as feedstock for production of the biopolymer polyhydroxybutyrate (PHB) by alphaproteobacterial methanotrophs. In vivo assessment of PHB production would shed light on the biosynthesis process and guide design of improved production strategies, but it is currently difficult to perform efficiently. In this study, the alphaproteobacterial methanotroph Methylocystis sp. Rockwell was grown on methane with three different nitrogen sources (ammonium, nitrate, and atmospheric nitrogen), and biomass samples were harvested at defined time points during lag, exponential, and stationary growth phases. PHB cell content was analyzed at these sampling points via a standard gas chromatography-flame ionization detector method, which requires hydrolysis of PHB and esterification of the resulting monomer under acidic conditions, and a novel, rapid, cost-effective approach based on fixation and staining of bacterial cells via Nile Blue A fluorescent dye enabling differential staining of cell membranes and intracellular PHB granules for single-cell analysis through fluorescence microscopy. Overall, the two PHB quantification approaches were in agreement at all stages of growth and in all three growing conditions tested. The PHB cell content was greatest with atmospheric nitrogen as a nitrogen source, followed by ammonium and nitrate. Under atmospheric nitrogen and ammonium conditions, PHB cell content decreased with growth progression, while under nitrate conditions PHB cell content remained unchanged in all growth phases. In addition to presenting a rapid, efficient method enabling in vivo quantification of PHB production, the present study highlights the impact of nitrogen source on PHB production by Methylocystis sp. Rockwell. KEY POINTS: • A novel fluorescence microscopy method to quantify PHB in single cells was developed • The microscopy method was validated by the derivation/gas chromatography method • Methylocystis sp. Rockwell synthesizes PHB granules without nutrient stress.

ToxR modulates biofilm formation in fish pathogen Vibrio harveyi

Vibrio harveyi is a common aquaculture pathogen causing diseases in a variety of aquatic animals. toxR, a conserved virulence-associated gene in vibrios, is identified in V. harveyi 345, a pathogenic strain isolated from diseased fish. In this study, to gain insight into function of ToxR in V. harveyi, an in-frame deletion of the toxR gene was constructed to reveal the role of ToxR in the physiology and virulence of V. harveyi. The statistical analysis showed no significant differences in the growth ability, motility, extracellular protease secretion, antibiotic susceptibility, virulence by intraperitoneal injection and the ability of V. harveyi to colonize the spleen and liver tissues of the pearl gentian grouper between the wild-type (WT) and the toxR mutant. However, the deletion of toxR increased the biofilm formation. The structure of the V. harveyi biofilm was further analysed by using scanning electron microscopy (SEM) and confocal laser scanning microscopy, and the results showed that deletion of toxR increased the number and density of V. harveyi biofilm. Since biofilm production is flagella, exopolysaccharide (EPS) and lipopolysaccharide dependent, 16 of V. harveyi biofilm-related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR, the expression levels of these genes, including genes flrB, motY and mshA, flaE, flrA and gmhD, were significantly up-regulated in the ΔtoxR+ strain as compared with the WT+ and C-ΔtoxR strains during the early and mid-exponential, while epsG, flaA, flaE, flgD, flgE, flrB, flrC, lpxB, motY, mshA and scrG genes were inhibited because of deletion of the toxR gene in the stationary growth phase. Our results indicate that ToxR plays an important role in controlling the biofilm in V. harveyi.

LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi.

Vibrio harveyi can cause infections and diseases in a variety of marine vertebrates and invertebrates, which are harmful to the aquaculture industry. The LuxS quorum-sensing system regulates the expression of virulence factors in a wide variety of pathogenic bacteria. In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. Statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and luxS mutant. However, deletion of luxS decreased the secretion of extracellular protease, while increasing swimming and swarming abilities. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented this defect. Since motility is flagella dependent, 16 V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly upregulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phases. Our results indicate that LuxS plays an important role in controlling motility, flagella biogenesis, and extracellular protease secretion in V. harveyi.

Comparative genomic identification and characterization of npcRNA homologs in Proteus vulgaris

Proteus vulgaris is a rod-shaped Gram-negative bacterium known to be the member of Enterobacteriaceae that is able to cause disease in human being. Generally, non-protein-coding RNAs (npcRNAs) do not code for proteins, but they play a vital role in gene regulation at the RNA level including pathogenicity. The present study aims at elucidating homologous npcRNAs from other bacteria in Proteus vulgaris. A comparative genomic analysis was carried out to identify npcRNA homolog of other Enterobacteriaceae pathogens in Proteus vulgaris. A total of 231 npcRNAs previously reported in Salmonella typhi, Salmonella typhimurium and Escherichia coli were screened using BLASTn tool against Proteus vulgaris genome. Interestingly, 33 npcRNAs are homologs to Proteus vulgaris. Northern blot analysis of 6 out of 33 npcRNA candidates confirmed their expression and showed that most of them are differentially expressed during lag, exponential and stationary growth phases. This study is the first approach of identification and characterization of npcRNAs in P. vulgaris. Hence, this could be a pioneer study to further validate the regulatory functions of these npcRNAs to fill the gaps in understanding of the pathogenicity of P. vulgaris.

DNA cytosine methylation at the lexA promoter of Escherichia coli is stationary phase specific.

The bacterial DNA damage response pathway (SOS response) is composed of a network of genes regulated by a single transcriptional repressor, LexA. The lexA promoter, itself, contains two LexA operators, enabling negative feedback. In Escherichia coli, the downstream operator contains a conserved DNA cytosine methyltransferase (Dcm) site that is predicted to be methylated to 5-methylcytosine (5mC) specifically during stationary phase growth, suggesting a regulatory role for DNA methylation in the SOS response. To test this, we quantified 5mC at the lexA locus, and then examined the effect of LexA on Dcm activity, as well as the impact of this 5mC mark on LexA binding, lexA transcription, and SOS response induction. We found that 5mC at the lexA promoter is specific to stationary phase growth, but that it does not affect lexA expression. Our data support a model where LexA binding at the promoter inhibits Dcm activity without an effect on the SOS regulon.

Two Novel Semisynthetic Lipoglycopeptides Active against Staphylococcus aureus Biofilms and Cells in Late Stationary Growth Phase.

The increase in antibiotic resistance among Gram-positive bacteria underscores the urgent need to develop new antibiotics. New antibiotics should target actively growing susceptible bacteria that are resistant to clinically accepted antibiotics including bacteria that are not growing or are protected in a biofilm environment. In this paper, we compare the in vitro activities of two new semisynthetic glycopeptide antibiotics, MA79 and ERJ390, with two clinically used glycopeptide antibiotics—vancomycin and teicoplanin. The new antibiotics effectively killed not only exponentially growing cells of Staphylococcus aureus, but also cells in the stationary growth phase and biofilm.

The Dynamics of Mycoplasma gallisepticum Nucleoid Structure at the Exponential and Stationary Growth Phases.

The structure and dynamics of bacterial nucleoids play important roles in regulating gene expression. Bacteria of class Mollicutes and, in particular, mycoplasmas feature extremely reduced genomes. They lack multiple structural proteins of the nucleoid, as well as regulators of gene expression. We studied the organization of Mycoplasma gallisepticum nucleoids in the stationary and exponential growth phases at the structural and protein levels. The growth phase transition results in the structural reorganization of M. gallisepticum nucleoid. In particular, it undergoes condensation and changes in the protein content. The observed changes corroborate with the previously identified global rearrangement of the transcriptional landscape in this bacterium during the growth phase transition. In addition, we identified that the glycolytic enzyme enolase functions as a nucleoid structural protein in this bacterium. It is capable of non-specific DNA binding and can form fibril-like complexes with DNA.

Homeophasic Adaptation in Response to UVA Radiation in Pseudomonas aeruginosa: Changes of Membrane Fatty Acid Composition and Induction of desA and desB Expression.

In bacteria, exposure to changes in environmental conditions can alter membrane fluidity, thereby affecting its essential functions in cell physiology. To adapt to these changes, bacteria maintain appropriate fluidity by varying the composition of the fatty acids of membrane phospholipids, a phenomenon known as homeophasic adaptation. In Pseudomonas aeruginosa, this response is achieved mainly by two mechanisms of fatty acid desaturation: the FabA-FabB and DesA-DesB systems. This study analyzed the effect of ultraviolet-A (UVA) radiation-the major fraction of solar UV radiation reaching the Earth's surface-on the homeophasic process. The prototypical strain PAO1 was grown under sublethal UVA doses or in the dark, and the profiles of membrane fatty acids were compared at early logarithmic, logarithmic and stationary growth phases. In the logarithmic growth phase, it was observed that growth under sublethal UVA doses induced the expression of the desaturase-encoding genes desA and desB and increased the proportion of unsaturated fatty acids; in addition, membrane fluidity could also increase, as suggested by the indices used as indicators of this parameter. The opposite effect was observed in the stationary growth phase. These results demonstrate the relevant role of UVA on the homeophasic response at transcriptional level.

Fis Connects Two Sensory Pathways, Quorum Sensing and Surface Sensing, to Control Motility in Vibrio parahaemolyticus.

Factor for inversion stimulation (Fis) is a global regulator that is highly expressed during exponential phase growth and undetectable in stationary phase growth. Quorum sensing (QS) is a global regulatory mechanism that controls gene expression in response to changes in cell density and growth phase. In Vibrio parahaemolyticus, a marine species and a significant human pathogen, the QS regulatory sRNAs, Qrr1 to Qrr5, are expressed during exponential growth and negatively regulate the high cell density QS master regulator OpaR. OpaR is a positive regulator of capsule polysaccharide (CPS) formation, which is required for biofilm formation, and is a repressor of lateral flagella required for swarming motility. In V. parahaemolyticus, we show that Fis is a positive regulator of the qrr sRNAs expression. In an in-frame fis deletion mutant, qrr expression was repressed and opaR expression was induced. The Δfis mutant produced CPS and biofilm, but swarming motility was abolished. Also, the fis deletion mutant was more sensitive to polymyxin B. Swarming motility requires expression of both the surface sensing scrABC operon and lateral flagella laf operon. Our data showed that in the Δfis mutant both laf and scrABC genes were repressed. Fis controlled swarming motility indirectly through the QS pathway and directly through the surface sensing pathway. To determine the effects of Fis on cellular metabolism, we performed in vitro growth competition assays, and found that Δfis was outcompeted by wild type in minimal media supplemented with intestinal mucus as a sole nutrient source. The data showed that Fis positively modulated mucus components L-arabinose, D-gluconate and N-acetyl-D-glucosamine catabolism gene expression. In an in vivo colonization competition assay, Δfis was outcompeted by wild type, indicating Fis is required for fitness. Overall, these data demonstrate a global regulatory role for Fis in V. parahaemolyticus that includes QS, motility, and metabolism.

Effect of the Concentration of Extracellular Polymeric Substances (EPS) and Aeration Intensity on Waste Glycerol Valorization by Docosahexaenoic Acid (DHA) Produced in Heterotrophic Culture of Schizochytrium sp

The study aimed to determine the effectiveness of docosahexaenoic acid (DHA) production by Schizochytrium sp. biomass fed with waste glycerol depending on the concentration of extracellular polymeric substances (EPS) in the culture medium and medium aeration effectiveness. The microalgae from the genus Schizochytrium sp. were proved to be capable of producing EPS composed of glucose, galactose, mannose, fucose, and xylose. The highest EPS concentration, reaching 8.73 ± 0.09 g/dm3, was determined at the stationary growth phase. A high EPS concentration caused culture medium viscosity to increase, contributing to diminished oxygen availability for cells, lower culture effectiveness, and reduced waste glycerol conversion to DHA. The Schizochytrium sp. culture variant found optimal in terms of the obtained technological effects and operating costs was performed at the volumetric oxygen mass transfer coefficient of kLa = 600 1/h, which enabled obtaining dry cell weight (DCW) of 147.89 ± 4.77 g/dm3, lipid concentration of 69.44 ± 0.76 g/dm3, and DHA concentration in the biomass reaching 29.44 ± 0.36 g/dm3. The effectiveness of waste glycerol consumption in this variant reached 3.76 ± 0.31 g/dm3·h and 3.16 ± 0.22 g/gDCW.

Novel RclSAR three-component system regulates expression of the intI1 gene in the stationary growth phase

The rapid and appropriate response of Pseudomonas spp. to environmental fluctuations has been enabled by numerous signal transduction regulatory systems. Regulatory systems in Pseudomonas aeruginosa are organized in a complex network which provides quick and fine-tuned cellular response through regulation of virulence and antibiotic resistance determinants production. Mobile integrons represent genetic elements included in the rapid dissemination of multiple antibiotic resistance determinants. The key factor of integron dynamics is enzyme integrase. So far, global regulators LexA, RpoS and PsrA have been recognized as regulators of the intI1 transcription. In this study, we discovered novel activator of the intI1 transcription, sensor kinase RclS, in Pseudomonas putida WCS358. This regulation is limited to stationary growth phase and appears to be indirect, at least through regulation of the rpoS expression. Sensor kinase RclS is a part of novel three-component system Rcl (Roc-like) together with two response regulators, RclR and RclA. RclS acted as a negative regulator of the rclA transcription, while the role in the rclR transcription regulation could not be defined. The RclSAR regulatory system seems to be a part of complex intI1 regulatory network which includes major stress response (SOS and RpoS) regulons.

PSXI-22 Study of biosorption mechanisms of movable forms of lead by soil isolates of the genus Bacillus in experiments in vitro

Abstract The research was supported by the Ministry of Science and Higher Education in accordance with the state assignment for Ural State Mining University No. 0833-2020-0008 ‘Development and environmental and economic substantiation of the technology for reclamation of land disturbed by the mining and metallurgical complex based on reclamation materials and fertilizers of a new type’. We obtain the scientific results by using funds of the Center for the collective use of scientific equipment of the Federal Scientific Center of biological systems and agricultural technologies of RAS as well (No Ross RU.0001.21 PF59, the Unified Russian Register of Centers for Collective Use - http://www.ckp-rf.ru/ckp/77384).The search for natural bioremediators to restore crop and grazing lands in conditions of the increased anthropogenic load is a relevant and promisingly significant area of research. Thus, we were tasked to assess the prospect of using the physiological and adaptive characteristics of microorganisms of the genus Bacillus as an active biosorbent of movable forms of lead from the substrate on the in vitro model.Soil isolates of microorganisms of the genus Bacillus extracted from samples from areas with increased lead levels were used to achieve our aim. Pb(NO3)2 solutions in the concentration range from 1 to 0.063M were used as the factor regulating the growth. Methods used in the work were: the method of agar basins, the nephelometric method, atomic-absorption spectroscopy, and atomic force microscopy. B. subtilis and B. cereus were isolated from soil samples. The maximum resistance to different concentrations of Pb(NO3)2 was revealed for B. subtilis. The presence of lead cations in the nutrient substrate significantly increased the population density to 26.0%. At the same time, the percentage of lead sorption in the initial stage of the stationary growth phase was 65.3% with the maximum localization on the surface structures of the cell membrane.

Modification of physico-chemical surface properties and growth of Staphylococcus aureus under hyperglycemia and ketoacidosis conditions

Diabetes is a widely spread disease affecting the quality of life of millions of people around the world and is associated to a higher risk of developing infections in different parts of the body. The reasons why diabetes enhances infection episodes are not entirely clear; in this study our aim was to explore the changes that one of the most frequently pathogenic bacteria undergoes when exposed to hyperglycemia and ketoacidosis conditions. Physical surface properties such as hydrophobicity and surface electrical charge are related to bacterial growth behavior and the ability of Staphylococcus aureus to form biofilms. The addition of glucose made bacteria more negatively charged and with moderate-intermediate hydrophobicity. Ketone bodies increased hydrophobicity to approximately 75% and pathological concentrations hindered some of the bacterial surface charge by decreasing the negative zeta potential of cells. When both components were present, the bacterial physical surface changes were more similar to those observed in ketone bodies, suggesting a preferential adsorption of ketone bodies over glucose because of the more favorable solubility of glucose in water.Glucose diabetic concentrations gave the highest number of bacteria in the stationary phase of growth and provoked an increase in the biofilm slime index of around 400% in relation to the control state. Also, this situation is related with an increase of bacterial coverage. The combination of a high concentration of glucose and ketone bodies, which corresponds to a poorly controlled diabetic situation, appears associated with an early infection phase; increased hydrophobic attractive force and reduced electrostatic repulsion between cells results in better packing of cells within the biofilm and more efficient retention to the host surface.Knowledge of bacterial response in high amount of glucose and ketoacidosis environments can serve as a basis for designing strategies to prevent bacterial adhesion, biofilm formation and, consequently, the development of infections.

Lipid and Fatty Acids Accumulation Features of Entomoneis cf. paludosa during Exponential and Stationary Growth Phases in Laboratory Culture

Diatoms are capable of accumulating substantial amounts of triacylglycerides in their cells, which differ in the composition of fatty acids depending on the conditions of cultivation, making them attractive subjects in biotechnology. In the present study, we characterized the structural features of lipid bodies in the diatom Entomoneis cf. paludosa (W. Smith) Reimer strain 8.0727-B and revealed the peculiarities of fatty acid composition in cultures during the stationary and exponential growth phases. Laser scanning confocal microscopy revealed an increased number of lipid bodies in the cytoplasm during the stationary phase of culture growth. Electron microscopy of ultrathin sections showed that an extreme increase in the number and size of plastoglobules in the cells occurs in the stationary phase of culture growth. The gas chromatography with mass spectrometric detection method revealed differences in the fatty acid composition depending on the growth phase. The studied strain can be recommended as a source of hexadecanoic and octadecanoic fatty acids from the culture during the stationary growth phase, as well as eicosapentaenoic fatty acid from the culture during the exponential growth phase.

Microbial synthesis and evaluation of bactericidal properties of cadmium sulfide nanoparticles

Introduction. The productivity of microbial synthesis of stable nanoparticles is determined by the growth stage of the populations of bacterial cultures used to obtain nanostructures. The study of the biocidal activity of biogenic nanoparticles of cadmium sulfide (NPsCdS), comparable in properties with nanomaterials obtained by physicochemical methods, is promising.The aim of this work was to evaluate the effect of the cell growth phase of the bacterial strains Bacillus subtilis 168 and Shewanella oneidensis MR-1 on the efficiency of biosynthesis of NPsCdS and to study their bactericidal properties against a number of gram-positive and gram-negative strains of microorganisms.Material and methods. Nanoparticles were obtained by introducing Na2S and CdCl2 salts to a final concentration of 2 mM : 2 mM in liquid bacterial cultures with cells in different phases of growth. The efficiency of NPsCdS biosynthesis was evaluated by the optical density of aqueous nanoparticles solutions. The bactericidal properties of NPsCdS were determined by the diameter of zone of inhibition growth of gram-positive bacteria B. subtilis 168, B. amyloliquefaciens, Streptococcus salivarius, Rhodococcus rhodochrous and gram-negative S. oneidensis MR-1, Escherichia coli K-12, Pseudomonas putida.Results. It was found that the use of cells in the stationary phase of growth (18–24 hours) contributes to obtaining the maximum amount of NPsCdS corresponding to concentrations of 1.0–1.2 mg/ml. The high antimicrobial activity of NPsCdS was shown against gram-positive microorganisms, among gram-negative bacteria, P. putida strain showed insignificant sensitivity.Discussion. The experimental results expand scientific data about the effect of the phase of bacterial growth cycle on biosynthesis of nanoparticles. The stationary phase of growth of B. subtilis 168, S. oneidensis MR-1 is optimal for obtaining of NPsCdS. For the first time, the cytotoxicity of NPsCdS/Shewanella against bacteria of various taxonomic groups was demonstrated.Conclusion. An effective method for obtaining extracellular NPsCdS using bacteria B. subtilis 168, S. oneidensis MR-1 in the stationary phase of growth has been developed. The biocidal activity of biogenic NPsCdS was shown, which allows to consider them as a new class of antimicrobial agents.

Phosphate stress triggers the conversion of glycerol into l-carnitine in Pseudomonas fluorescens

Glycerol, a by-product of the biofuel industry is transformed into l-carnitine when the soil microbe Pseudomonas fluorescens is cultured in a phosphate-limited mineral medium (LP). Although the biomass yield was similar to that recorded in phosphate-sufficient cultures (HP), the rate of growth was slower. Phosphate was completely consumed in the LP cultures while in the HP media, approximately 35 % of the initial phosphate was detected at stationary phase of growth. The enhanced production of α-ketoglutarate (KG) in HP cultures supplemented with manganese was recently reported (Alhasawi et al., 2017). l-carnitine appeared to be a prominent metabolite in the spent fluid while the soluble cellular-free extract was characterized with peaks attributable to lysine, γ-butyrobetaine (GB), acetate and succinate in the LP cultures. Upon incubation with glycerol and NH4Cl, the resting cells readily secreted l-carnitine and revealed the presence of such precursors like GB, lysine and methionine involved in the synthesis of this trimethylated moiety. Functional proteomic studies of select enzymes participating in tricarboxylic acid cycle (TCA), oxidative phosphorylation (OP), glyoxylate cycle and l-carnitine synthesis revealed a major metabolic reconfiguration evoked by phosphate stress. While isocitrate dehydrogenase-NAD+ dependent (ICDH-NAD+) and Complex I were markedly diminished, the activities of γ-butyrobetaine aldehyde dehydrogenase (GBADH) and l-carnitine dehydrogenase (CDH) were enhanced. Real-time quantitative polymerase chain reaction (RT-qPCR) analyses pointed to an increase in transcripts of the enzymes γ-butyrobetaine dioxygenase (bbox1), S-adenosylmethionine synthase (metK) and l-carnitine dehydrogenase (lcdH). The l-carnitine/γ-butyrobetaine antiporter (caiT) was enhanced more than 400-fold in the LP cultures compared to the HP controls. This metabolic reprogramming modulated by phosphate deprivation may provide an effective technology to transform glycerol, an industrial waste into valuable l-carnitine.