Early Exponential Growth Phase Research Articles

ZnO nanoparticle engineered Pseudomonas aeruginosa SOA1 towards enhanced Rhamnolipid production

Rhamnolipids, the potential biosurfactants, have widespread applications in pharmaceutics, food, and agriculture fields. However, its low biosynthesis efficiency limits surfactant industrialization. In this study, different nanoparticles (NPs) were immobilized over Pseudomonas aeruginosa (SOA1) and targeted towards increased rhamnolipids production by increasing the secretion (key step of rhamnolipids biosynthesis). It was observed that, upon adding ZnO NPs (4.0 g/L), rhamnolipids titer increased 4.87 times (from 4.1 to 19.98 g/L) in the 10 L bioreactor. This is due to an increment in the expression of the gene (present in the rhamnolipids biosynthesis process) with the introduction of ZnO NPs to SOA1. Further, with ZnO NPs, few rhamnolipid biosynthesis genes (rhlA and rhlB) were significantly upregulated. In contrast, the rhamnose biosynthetic genes (rmlA, rmlC, and algC) were down regulated during the early and late exponential growth phases as studied in Quantitative real-time PCR. The biosurfactant generation is regulated by quorum-sensing genes, which exhibit a hierarchical expression profile. Specifically, in the early log phase (ZnO NPs cultivation), the last gene was elevated, but both pqsE and rhlI were stimulated in the late log phase. This experiment paves a new path toward improving the biosynthesis of microbial metabolites via. NPs engineering.

Bacterial Metabolites and Particle Size Determine Cerium Oxide Nanomaterial Biotransformation.

Soil is a major receptor of manufactured nanomaterials (NMs) following unintentional releases or intentional uses. Ceria NMs have been shown to undergo biotransformation in plant and soil organisms with a partial Ce(IV) reduction into Ce(III), but the influence of environmentally widespread soil bacteria is poorly understood. We used high-energy resolution fluorescence-detected X-ray absorption spectroscopy (HERFD-XAS) with an unprecedented detection limit to assess Ce speciation in a model soil bacterium (Pseudomonas brassicacearum) exposed to CeO2 NMs of different sizes and shapes. The findings revealed that the CeO2 NM's size drives the biotransformation process. No biotransformation was observed for the 31 nm CeO2 NMs, contrary to 7 and 4 nm CeO2 NMs, with a Ce reduction of 64 ± 14% and 70 ± 15%, respectively. This major reduction appeared quickly, from the early exponential bacterial growth phase. Environmentally relevant organic acid metabolites secreted by Pseudomonas, especially in the rhizosphere, were investigated. The 2-keto-gluconic and citric acid metabolites alone were able to induce a significant reduction in 4 nm CeO2 NMs. The high biotransformation measured for <7 nm NMs would affect the fate of Ce in the soil and biota.

The Bacillus anthracis S-layer is an exoskeleton-like structure that imparts mechanical and osmotic stabilization to the cell wall.

Surface layers (S-layers) are 2D paracrystalline protein monolayers covering the cell envelope of many prokaryotes and archaea. Proposed functions include a role in cell support, as scaffolding structure, as molecular sieve, or as virulence factor. Bacillus anthracis holds two S-layers, composed of Sap or EA1, which interchange in early and late exponential growth phase. We previously found that acute disruption of B. anthracis Sap S-layer integrity, by means of nanobodies, results in severe morphological cell surface defects and cell collapse. Remarkably, this loss of function is due to the destruction of the Sap lattice structure rather than detachment of monomers from the cell surface. Here, we combine force nanoscopy and light microscopy observations to probe the contribution of the S-layer to the mechanical, structural, and functional properties of the cell envelope, which have been so far elusive. Our experiments reveal that cells with a compromised S-layer lattice show a decreased compressive stiffness and elastic modulus. Furthermore, we find that S-layer integrity is required to resist cell turgor under hypotonic conditions. These results present compelling experimental evidence indicating that the S-layers can serve as prokaryotic exoskeletons that support the cell wall in conferring rigidity and mechanical stability to bacterial cells.

Mechanism of high D-aspartate production in the lactic acid bacterium Latilactobacillus sp. strain WDN19.

D-Aspartate (D-Asp) is a useful compound for a semisynthetic antibiotic and has potentially beneficial effects on humans. Several lactic acid bacteria (LAB) species produce D-Asp as a component of cell wall peptidoglycan. We previously isolated a LAB strain (named strain WDN19) that can extracellularly produce a large amount of D-Asp. Here, we show the factors that contribute to high D-Asp production ability. Strain WDN19 was most closely related to Latilactobacillus curvatus. The D-Asp production ability of strain WDN19 in a rich medium was 13.7-fold higher than that of L. curvatus DSM 20019. A major part of D-Asp was synthesized from L-Asp contained in the medium by aspartate racemase (RacD). During their cultivation, the RacD activity in strain WDN19 was higher than in strain DSM 20019, especially much higher in the early exponential growth phase because of the higher racD transcription and the higher activity of RacD itself of strain WDN19. In a synthetic medium, the extracellular production of D,L-Asp was observed in strain WDN19 but not in strain DSM 20019. The addition of L-asparagine (L-Asn) to the medium increased and gave D,L-Asp production in strains WDN19 and DSM 20019, respectively, suggesting L-Asp synthesis by L-asparaginase (AsnA). The L-Asn uptake ability of the strains was similar, but the AsnA activity in the middle exponential and early stationary growth phases and intracellular D,L-Asp was much higher in strain WDN19. In their genome sequences, only an aspartate aminotransferase gene was found among L-Asp-metabolizing enzymes, except for RacD, but was disrupted in strain WDN19 by transposon insertion. These observations indicated that the high D-Asp production ability of strain WDN19 was mainly based on high RacD and AnsA activities and L-Asp supply. KEY POINTS: • Strain WDN19 was suggested to be a strain of Latilactobacillus curvatus. • Extracellular high d-Asp production ability was not a common feature of L. curvatus. • High d-Asp production was due to high RacD and AnsA activities and l-Asp supply.

Antimicrobial activity of bacteriocin produced by a new Latilactobacillus curvatus sp.LAB-3H isolated from traditional yogurt.

In recent years, the use of bacteriocin-producing Lactobacillus species has received much attention in different areas, including using as probiotics, food preservation, and as broad antimicrobial spectrum activity. In this study, a bacteriocin-producingLactobacillusstrain was isolated from traditional yogurt. The isolate was identified by morphological, biochemical, 16S rRNA analyses, and designated asLatilactobacillus curvatusLAB-3H. The primary antimicrobial activity of the isolate was evaluated onMicrococcus luteusPTCC 1408 by the agar gel diffusion method. The growth ofL. curvatusLAB-3H in MRS broth at 37°C and its antimicrobial activity againstLactobacillus caseiATCC 39,392 was evaluated. In batch culture analysis, bacteriocin production starts at the early exponential growth phase and continues to the middle of the stationary phase about (660 AU/ml) at 28h and pH 3.8. Bacteriocin produced byL. curvatusLAB-3H showed antibacterial activity against some selected foodborne pathogens, includingListeria monocytogenesPTCC1294,Bacillus cereusPTCC1857,Staphylococcus aureusPTCC1917, andEscherichia coliPTCC1276. For purification of bacteriocin, ammonium sulfate precipitation and cation exchange chromatography methods were used. The maximum antimicrobial activity observed was about 3985.15 AU/mg of protein, which was a 249.22-fold increase, and 5.21% yield compared with that in the cell-free supernatant. The molecular weight of bacteriocin was approximately 55kDa by SDS-PAGE. The obtained results in this study demonstrate that bacteriocin fromL. curvatusLAB-3H is a potential candidate for controlling microbial contaminations and can be used in different sectors, such as food industries.

Contrasting patterns of carbon cycling and dissolved organic matter processing in two phytoplankton–bacteria communities

Abstract. Microbial consumption of phytoplankton-derived organic carbon in the pelagic food web is an important component of the global C cycle. We studied C cycling in two phytoplankton–bacteria systems (non-axenic cultures of a dinoflagellate Apocalathium malmogiense and a cryptophyte Rhodomonas marina) in two complementary experiments. In the first experiment we grew phytoplankton and bacteria in nutrient-replete conditions and followed C processing at early exponential growth phase and twice later when the community had grown denser. Cell-specific primary production and total community respiration were up to 4 and 7 times higher, respectively, in the A. malmogiense treatments. Based on the optical signals, accumulating dissolved organic C (DOC) was degraded more in the R. marina treatments, and the rate of bacterial production to primary production was higher. Thus, the flow of C from phytoplankton to bacteria was relatively higher in R. marina treatments than in A. malmogiense treatments, which was further supported by faster 14C transfer from phytoplankton to bacterial biomass. In the second experiment we investigated consumption of the phytoplankton-derived DOC by bacteria. DOC consumption and transformation, bacterial production, and bacterial respiration were all higher in R. marina treatments. In both experiments A. malmogiense supported a bacterial community predominated by bacteria specialized in the utilization of less labile DOC (class Bacteroidia), whereas R. marina supported a community predominated by copiotrophic Alpha- and Gammaproteobacteria. Our findings suggest that large dinoflagellates cycle relatively more C between phytoplankton biomass and the inorganic C pool, whereas small cryptophytes direct relatively more C to the microbial loop.

Diversity of bioactive compound content across 71 genera of marine, freshwater, and terrestrial cyanobacteria

Cyanobacterial blooms have increased in frequency, distribution, and intensity due to climate change and anthropogenic nutrient input. The release of bioactive compounds accumulated in these blooms can affect the health of humans and the environment. The co-occurrence of bioactive metabolites is well-documented in bloom samples from marine and freshwater ecosystems, with fewer reports from unialgal isolates. Cyanobacteria also are important terrestrial ecosystem components, especially in drylands, but reports on bioactive molecules from terrestrial cyanobacteria are sparse. This study determined bioactive metabolite profiles for 71 genera of cyanobacteria from seven orders isolated from freshwater (12 genera), marine (15 genera), and terrestrial (44 genera) habitats originally. Cultures were harvested for bioactive metabolites when entering the late-exponential phase for all 157 strains, and 33 were sampled at both early and late exponential phases. Bioactive metabolites were analyzed using an ultra high performance/pressure liquid chromatography in-line with a time-of-flight mass spectrometer. Overall, 12 bioactive classes of the 28 identified were ubiquitous in all samples. On average, each freshwater genus produced ∼12 bioactive classes, whereas each marine genus contained > 4 bioactive classes, and each terrestrial genus contained ∼6 bioactive classes. While 10 of 12 freshwater genera produced at least 10 bioactive classes, only a single genus each from marine and terrestrial habitats had the same number of bioactive classed accumulated. Aeruginosin was found in 58 of 71 total genera, carmabin in 51 of 71 genera, and anabaenopeptin in 48 of 71 genera. Chemotaxonomic use of these secondary metabolites may help resolve higher-level genetic classification(s). An additional growth curve experiment showed that bioactive metabolites were produced at both early and late exponential growth phases. The bioactive metabolite accumulation pattern between early and late exponential phases differed by bioactive classes, genera, and habitats. This survey of 55 bioactive classes in cyanobacteria isolated from freshwater, marine, and terrestrial habitats (71 genera) provides as one of the first systematic bioactive metabolite profiles for cyanobacteria, which should be useful in environmental and drinking water management. Further, it offers novel insights about the toxin potential of selected terrestrial cyanobacteria.

Transcriptome-wide study in the green microalga Messastrum gracile SE-MC4 identifies prominent roles of photosynthetic integral membrane protein genes during exponential growth stage

The non-model microalga Messastrum gracile SE-MC4 is a potential species for biodiesel production. However, low biomass productivity hinders it from passing the life cycle assessment for biodiesel production. Therefore, the current study was aimed at uncovering the differences in the transcriptome profiles of the microalgae at early exponential and early stationary growth phases and dissecting the roles of specific differential expressed genes (DEGs) involved in cell division during M. gracile cultivation. The transcriptome analysis revealed that the photosynthetic integral membrane protein genes such as photosynthetic antenna protein were severely down-regulated during the stationary growth phase. In addition, the signaling pathways involving transcription, glyoxylate metabolism and carbon metabolism were also down-regulated during stationary growth phase. Current findings suggested that the coordination between photosynthetic integral membrane protein genes, signaling through transcription and carbon metabolism classified as prominent strategies during exponential growth stage. These findings can be applied in genetic improvement of M. gracile for biodiesel application.

Heavy Vacuum Gas Oil Upregulates the Rhamnosyltransferases and Quorum Sensing Cascades of Rhamnolipids Biosynthesis in Pseudomonas sp. AK6U.

We followed a comparative approach to investigate how heavy vacuum gas oil (HVGO) affects the expression of genes involved in biosurfactants biosynthesis and the composition of the rhamnolipid congeners in Pseudomonas sp. AK6U. HVGO stimulated biosurfactants production as indicated by the lower surface tension (26 mN/m) and higher yield (7.8 g/L) compared to a glucose culture (49.7 mN/m, 0.305 g/L). Quantitative real-time PCR showed that the biosurfactants production genes rhlA and rhlB were strongly upregulated in the HVGO culture during the early and late exponential growth phases. To the contrary, the rhamnose biosynthesis genes algC, rmlA and rmlC were downregulated in the HVGO culture. Genes of the quorum sensing systems which regulate biosurfactants biosynthesis exhibited a hierarchical expression profile. The lasI gene was strongly upregulated (20-fold) in the HVGO culture during the early log phase, whereas both rhlI and pqsE were upregulated during the late log phase. Rhamnolipid congener analysis using high-performance liquid chromatography-mass spectrometry revealed a much higher proportion (up to 69%) of the high-molecularweight homologue Rha–Rha–C10–C10 in the HVGO culture. The results shed light on the temporal and carbon source-mediated shifts in rhamonlipids’ composition and regulation of biosynthesis which can be potentially exploited to produce different rhamnolipid formulations tailored for specific applications.

Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius.

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC–gaa–glgX and amyA–glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC–gaa–glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

Monitoring of three-phase variations in the mortality of COVID-19 pandemic using control charts: where does Pakistan stand?

BackgroundAt the end of December 2019, the world in general and Wuhan, the industrial hub of China, in particular, experienced the COVID-19 pandemic. Massive increment of cases and deaths occurred in China and 209 countries in Europe, America, Australia, Asia and Pakistan. Pakistan was first hit by COVID-19 when a case was reported in Karachi on 26 February 2020. Several methods were presented to model the death rate due to the COVID-19 pandemic and to forecast the pinnacle of reported deaths. Still, these methods were not used in identifying the first day when Pakistan enters or exits the early exponential growth phase.ObjectiveThe present study intends to monitor variations in deaths and identify the growth phases such as pre-growth, growth, and post-growth phases in Pakistan due to the COVID-19 pandemic.MethodsNew approaches are needed that display the death patterns and signal an alarming situation so that corrective actions can be taken before the condition worsens. To meet this purpose, secondary data on daily reported deaths in Pakistan due to the COVID-19 pandemic have been considered. The n}{} c and exponentially weighted moving average (EWMA) control charts have been used for monitoring variations.ResultsThe n}{} c-chart shows that Pakistan switches from the pre-growth to the growth phase on 31 March 2020. The EWMA chart demonstrates that Pakistan remains in the growth phase from 31 March 2020 to 17 August 2020, with some indications signaling a decrease in deaths. It is found that Pakistan moved to a post-growth phase for a brief period from 27 July 2020 to 28 July 2020. Pakistan switches to re-growth phase with an alarm on 31/7/2020, right after the short-term post-growth phase. The number of deaths starts decreasing in August in that Pakistan may approach the post-growth phase shortly.ConclusionThis amalgamation of control charts illustrates a systematic implementation of the charts for government leaders and forefront medical teams to facilitate the rapid detection of daily reported deaths due to COVID-19. Besides government and public health officials, it is also the public’s responsibility to follow the enforced standard operating procedures as a temporary remedy of this pandemic in ensuring public safety while awaiting a suitable vaccine to be discovered.

Superoxide production rates and hemolytic activity linked to cellular growth phases in Chattonella species (Raphidophyceae) and Margalefidinium polykrikoides (Dinophyceae)

The increased occurrence of ichthyotoxic blooms of Chattonella and Margalefidinium species has led to mass fish kills causing multi-million dollar economic losses in diverse marine environments. Their ichthyotoxicity may be driven by a synergistic effect of diverse chemical compounds, of which little is understood in terms of production and biological activity. This study analyzed the growth rates, superoxide radical (O2●−) production rate, lipid peroxidation (TBARS) levels, and hemolytic activity of the ichthyotoxic raphidophytes Chattonella complex (C. var. marina, C. var. ovata, C. var. antiqua), C. subsalsa and the dinoflagellate Margalefidinium polykrikoides. Growth interactions between Chattonella and M. polykrikoides were also analyzed in bi-algal cultures. Chattonella strains exhibited higher growth rates than M. polykrikoides in monocultures. Highest O2●− production rate and TBARS levels in cells occurred in the early exponential growth phase, where M. polykrikoides showed higher values compared with Chattonella strains. Highest hemolytic activity associated to higher cell abundance was caused by M. polykrikoides followed by raphidophytes during the stationary and exponential growth phase, respectively. Of the three Chattonella strains, only C. subsalsa growth was inhibited when grown in bi-algal cultures with M. polykrikoides. In conclusion, this study supports an ecological linkage between cell growth conditions and a dynamic production of O2●− and/or hemolysins, which may mediate its ichthyotoxicity. However, the potential involvement in mass fish kills and ecological implications for local trophic webs remains to be further elucidated.

Human early mesenchymal stromal cells delivered on porous lightweight biodegradable polycaprolactone-based microcarriers result in improved cartilage formation

Porous lightweight polycaprolactone (LPCL)-based microcarriers (MC) can be used for large-scale production of human mesenchymal stromal cells (MSC) in stirred bioreactors. These biodegradable MC can serve in delivering MSC, which exhibit improved bone healing in a rat calvarial defect model. Therefore, this study aims to explore their use as scaffolds to create chondrogenically differentiated, MSC-laden LPCL MC constructs for cartilage formation in vitro and in vivo in a rabbit osteochondral defect model. In-vitro studies, defining critical parameters that resulted in efficient chondrogenic differentiation of MSC expanded on the surface of LPCL MC were performed. The optimized MSC-LPCL MC constructs were transplanted in a rabbit osteochondral defect model and evaluated after 5 months, with histological staining and scoring. Our in vitro studies demonstrate that about 4.7 × 103 porous LPCL MC seeded with 50 × 103 cells at early exponential growth phase (21% cell confluency) resulted in the best MSC-PCL MC construct, with efficient cell growth and chondrogenic differentiation, after 21 days. Implantation of chondrogenically differentiated MSC-LPCL MC constructs resulted in the best cartilage healing outcomes: (i) 75% of samples (6 out 8 defects) displaying good healing outcomes, (ii) the greatest histological score at 22 ± 7 and 26 ± 7 (without and with subchondral bone evaluation respectively), and (iii) the highest mean score for 8 out of 12 categories, as compared to other transplant groups. This is the first study demonstrating that chondrogenically differentiated MSC-LPCL MC constructs can induce the most efficient cartilage healing in rabbits with an osteochondral defect.

Programmed Proteolysis of Chemotaxis Proteins in Sinorhizobium meliloti: Features in the C-Terminal Region Control McpU Degradation.

Chemotaxis and motility are important traits that support bacterial survival in various ecological niches and in pathogenic and symbiotic host interaction. Chemotactic stimuli are sensed by chemoreceptors or methyl-accepting chemotaxis proteins (MCPs), which direct the swimming behavior of the bacterial cell. In this study, we present evidence that the cellular abundance of chemoreceptors in the plant symbiont Sinorhizobium meliloti can be altered by the addition of several to as few as one amino acid residues and by including common epitope tags such as 3×FLAG and 6×His at their C termini. To further dissect this phenomenon and its underlying molecular mechanism, we focused on a detailed analysis of the amino acid sensor McpU. Controlled proteolysis is important for the maintenance of an appropriate stoichiometry of chemoreceptors and between chemoreceptors and chemotactic signaling proteins, which is essential for an optimal chemotactic response. We hypothesized that enhanced stability is due to interference with protease binding, thus affecting proteolytic efficacy. Location of the protease recognition site was defined through McpU stability measurements in a series of deletion and amino acid substitution mutants. Deletions in the putative protease recognition site had similar effects on McpU abundance, as did extensions at the C terminus. Our results provide evidence that the programmed proteolysis of chemotaxis proteins in S. meliloti is cell cycle regulated. This posttranslational control, together with regulatory pathways on the transcriptional level, limits the chemotaxis machinery to the early exponential growth phase. Our study identified parallels to cell cycle-dependent processes during asymmetric cell division in Caulobacter crescentusIMPORTANCE The symbiotic bacterium Sinorhizobium meliloti contributes greatly to growth of the agriculturally valuable host plant alfalfa by fixing atmospheric nitrogen. Chemotaxis of S. meliloti cells toward alfalfa roots mediates this symbiosis. The present study establishes programmed proteolysis as a factor in the maintenance of the S. meliloti chemotaxis system. Knowledge about cell cycle-dependent, targeted, and selective proteolysis in S. meliloti is important to understand the molecular mechanisms of maintaining a suitable chemotaxis response. While the role of regulated protein turnover in the cell cycle progression of Caulobacter crescentus is well understood, these pathways are just beginning to be characterized in S. meliloti In addition, our study should alert about the cautionary use of epitope tags for protein quantification.

Human early mesenchymal stromal cells delivered on biodegradable polycaprolactone based microcarriers result in improved cartilage formation

Background & Aim Porous polycaprolactone (PCL)-based microcarriers (MC) can be used for large-scale production of human mesenchymal stromal cells (MSC) in stirred bioreactors. These biodegradable MC can deliver MSC, which improved bone healing in a rat calvarial defect model. Hence, this study aims to explore their use as scaffolds to create chondrogenically differentiated, MSC-laden PCL MC constructs for cartilage formation in vitro and in vivo in a rabbit endochondral defect model. Methods, Results & Conclusion Design: In vitro studies defining critical parameters that resulted in efficient chondrogenic differentiation of MSC expanded on the surface of PCL MC were performed. The optimized, critically-defined MSC-PCL MC constructs were transplanted in a rabbit endochondral defect model and evaluated after 5 months with histological staining and scoring. In vitro studies demonstrate that about 4.7 × 10 3 porous PCL MC seeded with 50 × 10 3 cells at early exponential growth phase (21% cell confluency) resulted in the best MSC-PCL MC construct with efficient cell growth and chondrogenic differentiation after 21 days. Implantation of chondrogenically differentiated MSC-PCL MC constructs resulted in the best cartilage healing outcomes: (i) 75% of samples displaying good healing outcomes, (ii) the greatest histological score at 22 ± 7 and 26 ± 7 (without and with subchondral bone evaluation respectively), and (iii) the highest mean score for 8 out of 12 categories, as compared to other transplant groups. This is the first study demonstrating that chondrogenically differentiated MSC-PCL MC constructs can induce the most efficient cartilage healing in rabbits with endochondral defect.

Inorganic Cadmium Detection Using a Fluorescent Whole-Cell Bacterial Bioreporter

Cadmium pollution has become a serious environmental issue due to its toxicity and frequent entrance into environment components such as soil, water, and air via many anthropogenic sources. Over the last two decades, whole-cell bacterial bioreporters have been acknowledged as bio-sentinels for the determination of toxic heavy metals. Herein a sensitive and quite specific bacterial bioreporter was developed to cope with the need for the rapid and simple determination of cadmium. The construction and characterization of a fluorescence-based whole-cell cadmium bioreporter strain, Escherichia coli MG1655 (pBR-PzntA), was described which is based on the expression of green fluorescent protein under the control of the zntA gene promoter of heavy metal resistance determinant. The developed bioreporter was able determine cadmium at 5 µg/L after 3.5 hours of induction in a defined medium while the cadmium detection limit was improved to 2 µg/L after 1.5 hours by the use of an inorganic phosphate-limiting defined medium. Drastic changes in cadmium sensitivity were obtained between bioreporter cells induced at different growth phases. The maximum fluorescence performance was obtained for early exponential growth phase cells. This cadmium bioreporter was found to be more sensitive and specific to cadmium ions than to a wide range of heavy metals and was sensitive to only cadmium at drinking water quality standard concentrations. These findings will lead to future studies including integration of the bioreporter cells into a portable device to assess bioavailable cadmium levels in environmental samples which will provide a rapid and practical field detection technique.

Naringenin Inhibition of the Pseudomonas aeruginosa Quorum Sensing Response Is Based on Its Time-Dependent Competition With N-(3-Oxo-dodecanoyl)-L-homoserine Lactone for LasR Binding.

Bacterial quorum sensing (QS) is a cell-to-cell communication system that governs the expression of a large set of genes involved in bacterial–host interactions, including the production of virulence factors. Conversely, the hosts can produce anti-QS compounds to impair virulence of bacterial pathogens. One of these inhibitors is the plant flavonoid naringenin, which impairs the production of QS-regulated Pseudomonas aeruginosa virulence factors. In the present work, we analyze the molecular basis for such inhibition. Our data indicate that naringenin produces its effect by directly binding the QS regulator LasR, hence competing with its physiological activator, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12-HSL). The in vitro analysis of LasR binding to its cognate target DNA showed that the capacity of naringenin to outcompete 3OC12-HSL, when the latter is previously bound to LasR, is low. By using an E. coli LasR-based biosensor strain, which does not produce 3OC12-HSL, we determined that the inhibition of LasR is more efficient when naringenin binds to nascent LasR than when this regulator is already activated through 3OC12-HSL binding. According to these findings, at early exponential growth phase, when the amount of 3OC12-HSL is low, naringenin should proficiently inhibit the P. aeruginosa QS response, whereas at later stages of growth, once 3OC12-HSL concentration reaches a threshold enough for binding LasR, naringenin would not efficiently inhibit the QS response. To test this hypothesis, we analyze the potential effect of naringenin over the QS response by adding naringenin to P. aeruginosa cultures at either time zero (early inhibition) or at stationary growth phase (late inhibition). In early inhibitory conditions, naringenin inhibited the expression of QS-regulated genes, as well as the production of the QS-regulated virulence factors, pyocyanin and elastase. Nevertheless, in late inhibitory conditions, the P. aeruginosa QS response was not inhibited by naringenin. Therefore, this time-dependent inhibition may compromise the efficiency of this flavonoid, which will be effective just when used against bacterial populations presenting low cellular densities, and highlight the importance of searching for QS inhibitors whose mechanism of action does not depend on the QS status of the population.

A novel mechanism of RyeA/SraC induction under acid stress

RyeA/SraC is a cis-encoded small RNA (sRNA), which act as an anti-toxin to RpoS-regulated RyeB toxin in Escherichia coli. Ectopic expression of RyeA was reported to diminish the RyeB accumulation by serving as a RNA trap. Lower abundance of RyeA in the early exponential growth phase turned out to be the outcome of its degradation by RNase BN/Z. In the current study, we show that RyeA is an acid stress inducible sRNA, and global stress responsive factor RpoS appeared to be inessential in RyeA induction. Although, ryeB-pphA dicistronic transcript at low pH condition was stimulated by ∼4-fold, however, RyeB population was found to be decreased by > 50% under the same condition by the decoy action of enhanced RyeA accumulation. Investigation of the mechanism of RyeA induceduction at low pH in the exponential phase, revealed that RNase BN/Z, which catabolizes RyeA in the exponential phase, appeared to be highly sensitive to low pH stress. Both mRNA and protein level of RNase BN transpired to be decreased to <10% of their initial population. The expression of RyeA under acid stress is regulated by a feed-forward mechanism to normalize the RyeB profusion.

Continuous nanosecond pulsed electric field treatments foster the upstream performance of Chlorella vulgaris-based biorefinery concepts

Nanosecond pulsed electric field treatment (nsPEF) is an innovative, technology-driven, and resource-efficient approach to foster the upstream performance of microalgae-based biorefinery concepts to transform microalgae into economic more viable raw materials for the biobased industry. A processing window applying three treatments of 100 ns, 5 Hz, and 10 kV cm−1 to industrially relevant phototrophic Chlorella vulgaris in the early exponential growth phase significantly increased biomass yields by up to 17.53 ± 10.46% (p = 3.18 × 10−5). Treatments had limited effects on the carbon and pigment contents, but the protein content was decreased. The longest possible pulse width (100 ns) resulted in the highest biomass yield indicating underlying working mechanisms of enhanced cell proliferation based on intracellular and plasma membrane-related effects. The applicability to eukaryotes and prokaryotes, such as C. vulgaris and cyanobacteria highlights the possible impacts of nsPEF across multiple domains of the biobased industry relying on single-cell-based value-chains.

Proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production

BackgroundRhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids.ResultsRhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose.ConclusionsThe results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.