Late Exponential Growth Phase Research Articles

Gas Chromatography-Ion Mobility Spectrometry Reveals Acetoin as a Biomarker for Carbapenemase-Producing Klebsiella pneumoniae.

BACKGROUND This study aimed to detect the volatile organic compound (VOC), 3-hydroxy-2-butanone (acetoin) using gas chromatography-ion mobility spectrometry (GC-IMS) in antimicrobial-resistant Klebsiella pneumoniae (K. pneumoniae) carbapenemase (KPC)-producing bacteria. MATERIAL AND METHODS Using stromal fluid of blood culture bottles (BacT/ALERT® SA) as the medium, 3-hydroxy-2-butanone (acetoin) released by K. pneumoniae during growth was detected using GC-IMS. The impact of imipenem (IPM) and carbapenemase inhibitors [avibactam sodium or pyridine-2,6-dicarboxylic acid (DPA)] on the emission of 3-hydroxy-2-butanone (acetoin) from various carbapenemase-producing K. pneumoniae was further investigated. Subsequently, VOCal software was used to generate a pseudo-3D plot of 3-hydroxy-2-butanone (acetoin), and the relative peak volumes were exported for data analysis. Standard strains served as references, and the findings were validated with clinical isolates. RESULTS The pattern of temporal changes in the 3-hydroxy-2-butanone (acetoin) release from K. pneumoniae in the absence of IPM was consistent with the growth curve. After the IPM addition, carbapenemase-positive strains released significantly higher contents of 3-hydroxy-2-butanone (acetoin) than carbapenemase-negative strains at the late exponential growth phase (T2). Notably, adding avibactam sodium significantly decreased the 3-hydroxy-2-butanone (acetoin) content released from the class A carbapenemase-producing strains as compared to the absence of the carbapenemase inhibitor. Conversely, adding DPA significantly decreased the 3-hydroxy-2-butanone (acetoin) content released from the class B carbapenemase-producing strains (both standard and clinical strains, all P<0.05). CONCLUSIONS This study demonstrated the potential of 3-hydroxy-2-butanone (acetoin) as a VOC biomarker for detecting carbapenemase-producing K. pneumoniae, as revealed by GC-IMS analysis.

Growth phase-dependent changes in conformational and physicochemical properties of bacterial surface biopolymers lead to changes in bacterial adhesion strength at nanoscale and macroscale

This study presents a detailed investigation into conformational, physicochemical and adhesive characteristics of biopolymers on the surfaces of Escherichia coli and potential probiotic Bacillus subtilis harvested from middle-exponential phase (mid-EP), late-exponential phase (late-EP) and early-stationary phase (early-SP) of growth. The lengths to which bacterial surface biopolymers extend (biopolymer brush lengths), densities of grafted bacterial surface biopolymers indicating the amounts of molecules covering the bacterial surfaces (biopolymer grafting densities), adhesion forces of bacterial surface biopolymers to the model inert surfaces of silicon nitride (Si3N4), and the pull-off distances of biopolymers from Si3N4 were measured in water by atomic force microscopy (AFM). The Weibull analysis of AFM adhesion data showed that as the culture aged, the adhesive bonds between Si3N4 AFM tips and surface molecules of E. coli harvested from the culture were broken with a higher applied force. However, the highest applied force to break the bonds was required for B. subtilis in late-EP, followed by those required for cells in early-SP and mid-EP, respectively. The results of a steric model fitting to AFM approach force-distance (FD) curves and analysis of the pull-off distances in the AFM retraction FD curves showed higher biopolymer grafting density for E. coli in early-SP and longer biopolymer brush layer for B. subtilis in late-EP, which were associated with stronger adhesion to Si3N4 in water. The results of thermodynamic adhesion energy calculations based on the Wu model showed that polar interaction energy dominated the bacterial adhesion at the macroscale, the strength of which varied as a function of the growth phase for both E. coli and B. subtilis. The growth phase-dependent variation in polar components of thermodynamic adhesion energies between the bacteria and Si3N4 in water was consistent with the growth phase-dependent variation in the bond strengths between the bacteria and Si3N4 in water as revealed by the Weibull analysis of AFM adhesion data. Therefore, information obtained by Weibull analysis of nanoscale AFM bacterial adhesion data can be used to predict macroscale bacterial adhesion to the model inert Si3N4 surface in water.

A growth phase analysis on the influence of light intensity on microalgal stress and potential biofuel production

It is urgent to find alternatives to fossil fuels to mitigate the effects of climate change. High light intensity during microalgal growth can promote lipid and carbohydrate accumulation (feedstocks to biofuels). However, more research needs to be done concerning the effect of this factor on microalgal productivity and biochemical composition from a growth phase point of view. This study evaluated the impact of high light intensities (291, 527 and 1107 μmol m−2 s−1) on Chlorella vulgaris growth in synthetic wastewater, nutrient assimilation and biochemical composition in late-exponential and stationary growth phases. Under the highest light intensity, a higher growth rate (0.54 ± 0.02 d−1) and maximum productivity (166 ± 13 mg/L d−1) were achieved. Also, nutrient removal efficiencies at the late-exponential phase reached 97.0 ± 0.2 % and 98.2 ± 0.1 % for nitrogen and phosphorus, respectively. Under all light intensities tested at the stationary phase, microalgae also achieved high nutrient removal efficiencies (>95 %). Extreme light intensity (1107 μmol m−2 s−1) boosted the accumulation of carbohydrates (30 ± 3 % w/w) and lipids (13.0 ± 0.4 % w/w). Under lower light intensities, the content of these compounds did not change significantly between growth phases. The chlorophyll content decreased with the increasing light intensity and increasing exposure time. Exposing C. vulgaris to high light intensity seems promising to simultaneously treat wastewater and boost lipid and carbohydrate accumulation, which can be used for biofuel production.

Exploring the Genomic Landscape of Bacillus paranthracis PUMB_17 as a Proficient Phosphatidylcholine-Specific Phospholipase C Producer.

Phospholipases find versatile applications across industries, including detergent production, food modification, pharmaceuticals (especially in drug delivery systems), and cell signaling research. In this study, we present a strain of Bacillus paranthracis for the first time, demonstrating significant potential in the production of phosphatidylcholine-specific phospholipase C (PC-PLC). The investigation thoroughly examines the B. paranthracis PUMB_17 strain, focusing on the activity of PC-PLC and its purification process. Notably, the PUMB_17 strain displays extracellular PC-PLC production with high specific activity during the late exponential growth phase. To unravel the genetic makeup of PUMB_17, we employed nanopore-based whole-genome sequencing and subsequently conducted a detailed genome annotation. The genome comprises a solitary circular chromosome spanning 5,250,970 bp, featuring a guanine-cytosine ratio of 35.49. Additionally, two plasmids of sizes 64,250 bp and 5845 bp were identified. The annotation analysis reveals the presence of 5328 genes, encompassing 5186 protein-coding sequences, and 142 RNA genes, including 39 rRNAs, 103 tRNAs, and 5 ncRNAs. The aim of this study was to make a comprehensive genomic exploration that promises to enhance our understanding of the previously understudied and recently documented capabilities of Bacillus paranthracis and to shed light on a potential use of the strain in the industrial production of PC-PLC.

B. subtilis Sec and Srp Systems Show Dynamic Adaptations to Different Conditions of Protein Secretion.

SecA is a widely conserved ATPase that drives the secretion of proteins across the cell membrane via the SecYEG translocon, while the SRP system is a key player in the insertion of membrane proteins via SecYEG. How SecA gains access to substrate proteins in Bacillus subtilis cells and copes with an increase in substrate availability during biotechnologically desired, high-level expression of secreted proteins is poorly understood. Using single molecule tracking, we found that SecA localization closely mimics that of ribosomes, and its molecule dynamics change similarly to those of ribosomes after inhibition of transcription or translation. These data suggest that B. subtilis SecA associates with signal peptides as they are synthesized at the ribosome, similar to the SRP system. In agreement with this, SecA is a largely mobile cytosolic protein; only a subset is statically associated with the cell membrane, i.e., likely with the Sec translocon. SecA dynamics were considerably different during the late exponential, transition, and stationary growth phases, revealing that single molecule dynamics considerably alter during different genetic programs in cells. During overproduction of a secretory protein, AmyE, SecA showed the strongest changes during the transition phase, i.e., where general protein secretion is high. To investigate whether the overproduction of AmyE also has an influence on other proteins that interact with SecYEG, we analyzed the dynamics of SecDF, YidC, and FtsY with and without AmyE overproduction. SecDF and YidC did not reveal considerable differences in single molecule dynamics during overexpression, while the SRP component FtsY changed markedly in its behavior and became more statically engaged. These findings indicate that the SRP pathway becomes involved in protein secretion upon an overload of proteins carrying a signal sequence. Thus, our data reveal high plasticity of the SecA and SRP systems in dealing with different needs for protein secretion.

Unravelling the impact of fat content on the microbial dynamics and spatial distribution of foodborne bacteria in tri-phasic viscoelastic 3D models

The aim of the current study is to develop and characterise novel complex multi-phase in vitro 3D models, for advanced microbiological studies. More specifically, we enriched our previously developed bi-phasic polysaccharide (Xanthan Gum)/protein (Whey Protein) 3D model with a fat phase (Sunflower Oil) at various concentrations, i.e., 10%, 20%, 40% and 60% (v/v), for better mimicry of the structural and biochemical composition of real food products. Rheological, textural, and physicochemical analysis as well as advanced microscopy imaging (including spatial mapping of the fat droplet distribution) of the new tri-phasic 3D models revealed their similarity to industrial food products (especially cheese products). Furthermore, microbial growth experiments of foodborne bacteria, i.e., Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Lactococcus lactis on the surface of the 3D models revealed very interesting results, regarding the growth dynamics and distribution of cells at colony level. More specifically, the size of the colonies formed on the surface of the 3D models, increased substantially for increasing fat concentrations, especially in mid- and late-exponential growth phases. Furthermore, colonies formed in proximity to fat were substantially larger as compared to the ones that were located far from the fat phase of the models. In terms of growth location, the majority of colonies were located on the protein/polysaccharide phase of the 3D models. All those differences at microscopic level, that can directly affect the bacterial response to decontamination treatments, were not captured by the macroscopic kinetics (growth dynamics), which were unaffected from changes in fat concentration. Our findings demonstrate the importance of developing structurally and biochemically complex 3D in vitro models (for closer proximity to industrial products), as well as the necessity of conducting multi-level microbial analyses, to better understand and predict the bacterial behaviour in relation to their biochemical and structural environment. Such studies in advanced 3D environments can assist a better/more accurate design of industrial antimicrobial processes, ultimately, improving food safety.

Effect of carbon and nitrogen concentrations on lipid accumulation and regulation of acetyl-CoA carboxylase in Papiliotrema laurentii.

Biodiesel is an interesting alternative to petroleum diesel as it is renewable, biodegradable, and has a low pollutant content. Yeast oils can be used for biodiesel production instead of edible oils, mitigating the use of arable land and water for biodiesel production. Maximum lipid accumulation is reached at 48h of cultivation by the oleaginous yeast Papiliotrema laurentii UFV-1. Nevertheless, the effects of carbon and nitrogen concentrations on lipid accumulation, as well as the regulation of lipid metabolism in this yeast are still not well-characterised. Therefore, this work evaluated the effects of carbon and nitrogen concentrations on the lipid accumulation in P. laurentti, the expression of the ACC gene, and the activity of the enzyme acetyl-CoA carboxylase (ACCase) in different carbon:nitrogen ratios (C:N) and glucose concentrations. The variation of ammonium sulfate concentration did not affect the growth and lipid accumulation in P. laurentii UFV-1. On the other hand, glucose concentration remarkably influenced biomass and lipid production by this yeast. Therefore, the carbon concentration is more important than the nitrogen concentration for lipid production by P. laurentii UFV-1. Importantly, the levels of both ACC gene expression and ACCase activity were maximum during the late-exponential growth phase and decreased after reaching the highest lipid contents, which was easier evidenced during the accumulation and maximum lipid levels. As such, the reduction of ACCase enzyme activity seems to be related to the decrease in the expression level of the ACC gene.

Effect of NaCl stress on exoproteome profiles of Bacillus amyloliquefaciens EB2003A and Lactobacillus helveticus EL2006H.

Salt stress can affect survival, multiplication and ability of plant growth promoting microorganisms to enhance plant growth. Changes in a microbe's proteome profile is one of the mechanisms employed by PGPM to enhance tolerance of salt stress. This study was focused on understanding changes in the exoproteome profile of Bacillus amyloliquefaciens EB2003A and Lactobacillus helveticus EL2006H when exposed to salt stress. The strains were cultured in 100 mL M13 (B. amyloliquefaciens) and 100 mL De man, Rogosa and Sharpe (MRS) (L. helveticus) media, supplemented with 200 and 0 mM NaCl (control), at pH 7.0. The strains were then incubated for 48 h (late exponential growth phase), at 120 rpm and 30 (B. amyloliquefaciens) and 37 (L. helveticus) °C. The microbial cultures were then centrifuged and filtered sterilized, to obtain cell free supernatants whose proteome profiles were studied using LC-MS/MS analysis and quantified using scaffold. Results of the study revealed that treatment with 200 mM NaCl negatively affected the quantity of identified proteins in comparison to the control, for both strains. There was upregulation and downregulation of some proteins, even up to 100%, which resulted in identification of proteins significantly unique between the control or 200 mM NaCl (p ≤ 0.05), for both microbial species. Proteins unique to 200 mM NaCl were mostly those involved in cell wall metabolism, substrate transport, oxidative stress tolerance, gene expression and DNA replication and repair. Some of the identified unique proteins have also been reported to enhance plant growth. In conclusion, based on the results of the work described here, PGPM alter their exoproteome profile when exposed to salt stress, potentially upregulating proteins that enhance their tolerance to this stress.

Continuous production of an antimicrobial metabolite with the terrestrial cyanobacterium Chroococcidiopsis cubana

The terrestrial cyanobacterium Chroococcidiopsis cubana was identified as a producer of an antimicrobial substance inhibiting growth of the Gram-positive bacterium Micrococcus luteus and the yeast Candida auris. It was found that the production is initiated by nitrogen limitation, usually occurring during late exponential growth phase of the cyanobacterium. This effect was used for implementation of a continuous production set-up, which led to a significantly increased formation of the antimicrobial metabolite. The produced bioactive culture supernatant was purified by reverse phase chromatography; the most potent fraction caused a growth inhibition of over 90 % for both indicator organisms. The metabolite remained active until a temperature of approx. 45 °C; at increasing temperatures, the inhibiting effect decreased significantly. By identification of the trigger initiating the compound synthesis, a first scale-up could be implemented, leading to a reliable production of the antimicrobial metabolite. As Candida auris is an emerging pathogen causing serious infections, this work can be the first step to the development of a potent antifungal drug.

Spatiotemporal regulation of the BarA/UvrY two-component signaling system

The BarA/UvrY two-component signal transduction system mediates adaptive responses of Escherichia coli to changes in growth stage. At late exponential growth phase, the BarA sensor kinase autophosphorylates and transphosphorylates UvrY, which activates transcription of the CsrB and CsrC noncoding RNAs. CsrB and CsrC, in turn, sequester and antagonize the RNA binding protein CsrA, which posttranscriptionally regulates translation and/or stability of its target mRNAs. Here, we provide evidence that during stationary phase of growth, the HflKC complex recruits BarA to the poles of the cells and silences its kinase activity. Moreover, we show that during the exponential phase of growth, CsrA inhibits hflK and hflC expression, thereby enabling BarA activation upon encountering its stimulus. Thus, in addition to temporal control of BarA activity, spatial regulation is demonstrated.

Screening the Pollution-Tolerant Chlorococcum sp. (Chlorophyceae) Grown in Municipal Wastewater for Simultaneous Nutrient Removal and Biodiesel Production

Over the last few years and with increasing global climatic change, the international energy crisis and shortage of freshwater resources have raised many inquiries about global water security and energy. Therefore, finding out alternative and sustainable energy sources has become an important universal requirement. Here, we assessed the viability of exploiting municipal wastewater (WW) as a nutrient-rich growth medium for cultivating the pollution-tolerant coccoid green microalga Chlorococcum sp. (Chlorophyceae) to simultaneously remove nutrients and produce biodiesel. Chlorococcum sp. was isolated from municipal wastewater sampled from Menoufia Governorate, Egypt. Under the standard growth conditions and until reaching the late exponential growth phase, it was cultivated at different concentrations (25%, 50%, 75%, and 100%) of the secondary treated WW, and the findings were compared to the control (grown in BBM). The study results revealed that the 50% WW treatment was the most suitable approach for removing NO3−, NH4+, and TP with percentages of 96.9%, 98.4%, and 90.1%, respectively. Moreover, the 50% WW treatment produced the highest algal biomass (1.97 g L−1) and productivity (82 mg L−1 day−1). In addition, it showed the highest lipid production (600 mg L−1), with 25 mg L−1 day−1 lipid productivity and lipid yield with 30.5% of the cell dry weight (CDW). The gas chromatography–mass spectrometry (GC-MS) technique was applied to characterize fatty acid profiling, and it was found that oleic (C18:1) and palmitic (C16:0) fatty acids were present in much higher concentrations in Chlorococcum sp. cells grown in 50% WW as compared to the control, i.e., 44.43% and 27.38% vs. 36.75% and 21.36%, respectively. No big difference was present in linoleic (C18:2) fatty acid concentrations. Importantly, the biodiesel properties of our Chlorococcum sp. grown in 50% WW were consistent with the international biodiesel standards. In light of our findings, Chlorococcum sp. has a great potential for utilization as a biodiesel feedstock and for bioremediation of wastewater.

Investigation of factors in improving Agrobacterium-mediated gene transfer in Ruellia tuberosa L. and evaluation of α-glucosidase inhibitory activity in established hairy roots

Ruellia tuberosa (family Acanthaceae) is widely known in traditional medicine in Asian countries for the treatment of diabetes and other diseases. Its roots were demonstrated to possess a hypoglycemic ability in diabetic animal models. In this study, an original induced procedure was investigated to establish hairy root (HR) from R. tuberosa. With the aim of increasing the transformation rate, some induced factors (acetosyringone (AS) dosage, type of explant, age, infection time, bacterial density, co-cultivation duration) were individually examined. As a result, an improved procedure was implemented: ten-day-old in vitro cotyledon explants were injured and then immersed in the bacterial suspension (OD600 nm = 0.4) added 200 µM AS during 10 min. The infected explants were co-cultivated for 4 days in the Murashige &amp; Skoog (MS) medium before transferring to the medium containing cefotaxime for bacterial elimination. After thirty days of culture, the improved procedure revealed a synergistic effect by enhancing the rooting rate and number of secondary roots per explant up to 4.4- and 8.0-fold, respectively, in comparison with the original procedure. The R. tuberosa HR was then cultured in liquid MS medium and achieved the highest biomass production at the late exponential growth phase (3rd week). Its ethanol extract was also higher 2.0-fold in α-glucosidase inhibitory activity than that of the natural root. In conclusion, the α-glucosidase inhibitory activity of HR inducing by the improved procedure may offer an effective and reliable substitute for the utilization of this herbal plant.

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.

Rhodococcus oxybenzonivorans sp. nov., a benzophenone-3-degrading bacterium, isolated from stream sediment.

A Gram-stain-positive, facultative aerobic, oxidase-negative, catalase-positive, non-sporulating, and non-motile bacterium, which degraded benzophenone-3, was isolated from stream sediment collected in the Republic of Korea and designated as strain S2-17T. Cells of this strain were rod-shaped during the early growth phase but became coccoid after the late exponential growth phase. Bacterial growth was observed at 15-37 °C (optimum, 25-30 °C) and pH 6.0-9.5 (optimum, pH 7.5-8.5) and in the presence of 0-9.0 % (w/v) NaCl (optimum, 0-1.0 %). Menaquinone-8 (H2) was the sole isoprenoid quinone, and C16 : 0, C17 : 1 ω8c, summed feature 3 (comprising C16 : 1 ω7c/C16 : 1 ω6c) and C18 : 1 ω9c were the major fatty acids. The cell wall of strain S2-17T contained meso-diaminopimelic acid, and arabinose, galactose and mycolic acid were found in whole-cell hydrolysates, suggesting a chemotype IV cell wall. The G+C content of the genome was 65.6 mol%. Phylogenetic analyses revealed that strain S2-17T formed a phyletic lineage within the genus Rhodococcus and was most closely related to Rhodococcus jostii DSM 44719T (99.2 % 16S rRNA gene sequence similarity). Average nucleotide identity and digital DNA-DNA hybridization values between strain S2-17T and R. jostii DSM 44719T were 82.6 and 26.5 %, respectively, indicating differences between the species. Based on its phenotypic, chemotaxonomic and molecular features, strain S2-17T represents a novel species of the genus Rhodococcus, for which the name Rhodococcus oxybenzonivorans sp. nov. is proposed. The type strain is S2-17T (=KACC 19281T=JCM 32046T).

Antimicrobial Potentials of Bacillus Subtilis Isolated from Soil and Water

In this study, an attempt was made to extract antimicrobial substance from the soil isolate of Bacillus subtilis and explore its antimicrobial activity on some selected bacteria. The bacteria were typed using RAPD-PCR method to identify organism to strain level, using 16sr RNA A1A13 specific primers. Results revealed bands of different sizes identical in both strains of Bacillus subtilis. The antimicrobial potential of the soil isolate was determined by disc diffusion assay. The production of antimicrobial compound was recorded maximum at the late exponential growth phase. The filtered concentrate of the crude extract was used to determine antimicrobial activity on some selected gram positive and gram – negative bacteria. Partial purification of the crude extract was carried out using High pressure Liquid chromatography (HPLC), Zymogram analysis and TLC were performed to determine the preliminary biochemical nature. The molecular weight of the antimicrobial peptide was determined to be less than 2.5k Da. The presence of antibiotic Bacilysocin was discovered from the extract. The minimum inhibitory concentration MIC of the purified fraction showed (MIC) ranging from 0.5 to 10µg/ml for Staphylococcus aureus and Streptococcus pyogenes. The MIC for the gram–negative was 0ug/ml. The crude extracts showed antimicrobial activity only on gram positive selected bacteria strains, while the purified extract showed antimicrobial activity on both the gram-positive and gram-negative with more activity on the gram-positive. The zones of inhibition for crude extract 1 on Staphylococcus auerus were 8.0mm, 5.0mm, 3.0mm, 1.0mm; extract 2: 15.0mm, 10.0mm, 9.0mm, 7.0mm, and 6.0mm, 4.0mm, 3.0mm, 1.0mm respectively for extract 3. for Streptococcus pyogenes the zones of inhibition range from 2.0mm to 9.0mm, for extracts 1 and 2:10.0mm to 3.0m and10.0mm to 1.0mm for extract 3.for the gram negative test organisms, the zones of inhibition were 0.0mm for all the extracts tested against it.

Bacterial community associated with Pyrodinium bahamense var. compressum during late exponential growth phase based on 16S rRNA gene sequence analysis

Bacterial community associated with Pyrodinium bahamense var. compressum during late exponential growth phase based on 16S rRNA gene sequence analysis

Growth-phase specific regulation of cviI/R based quorum sensing associated virulence factors in Chromobacterium violaceum by linalool, a monoterpenoid.

Quorum sensing (QS)-dependent gene regulation in bacteria performs a vital role in synchronization of cell-density-dependent functions. In Chromobacterium violaceum QS-dependent cviI/R regulatory genes are activated during the mid- or late-exponential phase of growth. However, sufficient evidence is lacking on the role of QS inhibitors on gene regulation at different phases of growth. Hence, we report the role of linalool, a natural monoterpenoid on QS mediated gene regulation at different stages of growth in C. violaceum by performing biosensor, growth kinetic and gene expression studies. In vitro and in vivo studies were performed for establishing role of linalool in reducing the virulence and infection by using HEK-293T cell lines and Caenorhabditis elegans models respectively. C. violaceum CV026 with C6-HSL was used as control. The results showed linalool to be a QS inhibitor with an estimated IC50 of 63µg/mL for violacein inhibition. At this concentration the cell density difference (delta OD600) of 0.14 from the compound was observed indicating the quorum concentration. The expression of cviI/R was initiated at mid-log phase (~ 18h) and reached the maximum at 36h in control whereas in treatment it remained significantly downregulated at all time points. The expression of violacein biosynthetic genes vioA, vioC, vioD and vioE was also downregulated by linalool. Infection studies with linalool showed higher survival rates in HEK-293T cell lines and C. elegans compared to the infection control. Taken together, this study proves linalool to be a QS inhibitor capable of attenuation of QS by controlling the cell density through cviI/R downregulation at the early phase of growth and hence offering scope for its application for controlling infections.

A semi high-throughput method for real-time monitoring of curli producing Salmonella biofilms on air-solid interfaces

Biofilms enable bacteria to colonize numerous ecological niches. Bacteria within a biofilm are protected by the extracellular matrix (ECM), of which the fibril-forming amyloid protein curli and polysaccharide cellulose are major components in members of Salmonella, Eschericha and Mycobacterium genus. A shortage of real-time detection methods has limited our understanding of how ECM production contributes to biofilm formation and pathogenicity. Here we present optotracing as a new semi-high throughput method for dynamic monitoring of Salmonella biofilm growth on air-solid interfaces. We show how an optotracer with binding-induced fluorescence acts as a dynamic fluorescent reporter of curli expression during biofilm formation on agar. Using spectrophotometry and microscopic imaging of fluorescence, we analyse in real-time the development of the curli architecture in relation to bacterial cells. With exceptional spatial and temporal precision, this revealed a well-structured, non-uniform distribution of curli organised in distally projecting radial channel patterns. Dynamic monitoring of the biofilm also showed defined regions undergoing different growth phases. ECM structures were found to assemble in regions of late exponential growth phase, suggesting that ECM forms on site after bacteria colonize the surface. As the optotracer biofilm method expedites screening of curli production, providing exceptional spatial-temporal understanding of the surface-associated biofilm lifestyle, this method adds a new technique to further our understanding of bacterial biofilms.

Disclosing Lactobacillus delbrueckii subsp. bulgaricus intraspecific diversity in exopolysaccharides production

Exopolysaccharides production by 3 ropy strains of Lactobacillus delbrueckii subsp. bulgaricus of dairy origin was evaluated in synthetic medium by combining different approaches: impedometric measurements, fluorescent microscopy and flow cytometry analyses. The evaluation of ΔE by impedometric measurement (E%max-E%40h) allowed the detection of EPS production in synthetic medium, but the differences in EPS production kinetic was highlighted by flow cytometry analysis and fluorescent microcopy. This approach enabled us to unravel the diversity in EPS synthesis and release into the laboratory medium during the growth of the strains. Our results showed that the maximum EPS production occurred after 8 h of incubation, when cells were in late exponential growth phase. Furthermore, flow cytometry analysis revealed that only part of the cell population could be identified as EPS producer or as EPS-bounded cell. Therefore, the combined approach used, allowed us to define at the same time the kinetics of EPS production and release by three strains belonging to the same species and, highlight that the production of EPS depends also on the number of EPS-producing cells within the same population. This approach could be useful for the selection of strains to be used as starter cultures in dairy products where EPS production is considered an important feature.

Superoxide Production by the Red Tide-Producing Chattonella marina Complex (Raphidophyceae) Correlates with Toxicity to Aquacultured Fishes.

The marine raphidophyte Chattonella marina complex forms red tides, causing heavy mortalities of aquacultured fishes in temperate coastal waters worldwide. The mechanism for Chattonella fish mortality remains unresolved. Although several toxic chemicals have been proposed as responsible for fish mortality, the cause is still unclear. In this study, we performed toxicity bioassays with red sea bream and yellowtail. We also measured biological parameters potentially related to ichthyotoxicity, such as cell size, superoxide (O2•−) production, and compositions of fatty acids and sugars, in up to eight Chattonella strains to investigate possible correlations with toxicity. There were significant differences in moribundity rates of fish and in all biological parameters among strains. One strain displayed no ichthyotoxicity even at high cell densities. Strains were categorized into three groups based on cell length, but this classification did not significantly correlate with ichthyotoxicity. O2•− production differed by a factor of more than 13 between strains at the late exponential growth phase. O2•− production was significantly correlated with ichthyotoxicity. Differences in fatty acid and sugar contents were not related to ichthyotoxicity. Our study supports the hypothesis that superoxide can directly or indirectly play an important role in the Chattonella-related mortality of aquacultured fishes.