Middle Exponential Phase Research Articles

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.

New Insights into the Biological Functions of Essential TsaB/YeaZ Protein in Staphylococcus aureus.

TsaB/YeaZ represents a promising target for novel antibacterial agents due to its indispensable role in bacterial survival, high conservation within bacterial species, and absence of eukaryotic homologs. Previous studies have elucidated the role of the essential staphylococcal protein, TsaB/YeaZ, in binding DNA to mediate the transcription of the ilv-leu operon, responsible for encoding key enzymes involved in the biosynthesis of branched-chain amino acids-namely isoleucine, leucine, and valine (ILV). However, the regulation of ILV biosynthesis does not account for the essentiality of TsaB/YeaZ for bacterial growth. In this study, we investigated the impact of TsaB/YeaZ depletion on bacterial morphology and gene expression profiles using electron microscopy and deep transcriptomic analysis, respectively. Our results revealed significant alterations in bacterial size and surface smoothness upon TsaB/YeaZ depletion. Furthermore, we pinpointed specific genes and enriched biological pathways significantly affected by TsaB/YeaZ during the early and middle exponential phases and early stationary phases of growth. Crucially, our research uncovered a regulatory role for TsaB/YeaZ in bacterial autolysis. These discoveries offer fresh insights into the multifaceted biological functions of TsaB/YeaZ within S. aureus.

RNA Interference of Phenoloxidases of the Fall Armyworm, Spodoptera frugiperda, Enhance Susceptibility to Bacillus thuringiensis Protein Vip3Aa19.

Phenoloxidase (PO) is an important enzyme in the cellular immune system and is involved in defense against a wide range of pathogens, including Bacillus thuringiensis. Vip3Aa19 is secreted and expressed by Bacillus thuringiensis (Bt) at the middle exponential growth phase and is a kind of protein with efficient insecticidal activity against Spodoptera frugiperda. However, immune responses of the target insects have been regarded as a hindrance to Bt pathogenicity. This paper reports two phenoloxidase (PO) genes (SfPAE and SfPO2) identified from the hemocyte transcriptome data of the fall armyworm, Spodoptera frugiperda. qRT-PCR validation results showed that the expression levels of two PO genes were significantly upregulated after Vip3Aa19 (LC50 = 4.98 µg/g) toxin treatment compared with those of S. frugiperda fed an insecticide-free artificial diet. Meanwhile, two PO genes were expressed from the egg to adult stages even without an immune challenge. We noticed that at all developmental stages investigated in the S. frugiperda, SfPAE was generally expressed at a higher level than SfPO2. However, after Vip3Aa19 treatment, the SfPO2 gene mRNA expression level was significantly elevated in response to the toxin challenge. An injection of a specific double-stranded RNA (dsRNA) against POs could suppress its expression. The third instar larvae of S. frugiperda treated with dsRNA were much more susceptible to Vip3Aa19 toxin than the control larvae were. Notably, the mortality rate was nearly 90% after a dsPO2 injection. These results proved that SfPO2 was more important for the survival of S. frugiperda. Finally, RNA interference and then PO activity detection revealed that PO genes mainly existed in the hemolymph and played an important role in immune defense against Bt toxin.

Experimental validation of stability and applicability of Start Growth Time method for high-throughput bacterial ecotoxicity assessment.

Ecotoxicity assessments based on bacteria as model organisms are widely used for routine toxicity screening because it has the advantages of time-saving, high sensitivity, cost-effectiveness, and less ethical responsibility. Determination of ecotoxicity effect via bacterial growth can avoid the restriction of model bacteria selection and unique equipment requirements, but traditional viable cell count methods are relatively labor- and time-intensive. The Start Growth Time method (SGT) is a high-throughput and time-conserving method to determine the amount of viable bacterial cells. However, its usability and stability for ecotoxicity assessment are rarely studied. This study confirmed its applicability in terms of bacterial types (gram-positive and gram-negative), growth phases (middle exponential and early stationary phases), and simultaneous existence of dead cells (adjustment by flow cytometry). Our results verified that the stability of establishing SGT correlation is independent of the bacterial type and dead-cell portion. Moreover, we only observed the effect of growth phases on the slope value of established SGT correlation in Shewanella oneidensis, which suggests that preparing inoculum for the SGT method should be consistent in keeping its stability. Our results also elucidate that the SGT values and the live cell percentages meet the non-linear exponential correlation with high correlation coefficients from 0.97 to 0.99 for all the examined bacteria. The non-linear exponential correlation facilitates the application of the SGT method in the ecotoxicity assessment. Finally, applying the exponential SGT correlation to evaluate the ecotoxicity effect of copper ions on E. coli was experimentally validated. The SGT-based method would require about 6 to 7h to finish the assessment and obtain an estimated EC50 at 2.27 ± 0.04mM. This study demonstrates that the exponential SGT correlation can be a high-throughput, time-conversing, and wide-applicable method for bacterial ecotoxicity assessment.

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.

Dual use of a local Protosiphon isolate BENHA2020 for biodiesel production and antioxidant activity of lipid-free biomass: A novel biorefinery approach for biomass valorization

The present study aimed to isolate and evaluate a local Protosiphon sp. for biodiesel production and antioxidant activity of its residual lipid-free biomass. Phylogenetic analysis confirmed that the isolate is Protosiphon botryoides. In the control medium using 250 mg L−1 NaNO3 as a nitrogen source, cells showed 37.7 dw% lipid content at stationary phase. At different nitrogen levels, increasing of nitrogen concentration resulted in obvious increase in growth, showing the maximum biomass productivity of 0.133 g L−1 d−1 at middle exponential phase using 750 mg L−1 NaNO3, with simultaneous reduction in lipid content, reaching its lowest recorded value of 27.9 dw%. Due to enhancement of lipid content of N-deficient culture (125 mg L−1 NaNO3), it showed the highest lipid productivity of 41.3 mg L−1 d−1. Fatty acid analysis confirmed the dominance of linoleic acid, eicosatrienoic acid, oleic acid, and palmitic acid. Biodiesel characteristics complied with the international standards and showed better properties than some other reported biodiesel promising microalgae. In addition, lipid-free biomass showed high radical scavenging activity of 55.3%, which confirms its suitability as additional nutritional and pharmaceutical material for enhanced biomass valorization.

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.

Selective Biosynthesis of Furoic Acid From Furfural by Pseudomonas Putida and Identification of Molybdate Transporter Involvement in Furfural Oxidation.

Upgrading of furanic aldehydes to their corresponding furancarboxylic acids has received considerable interest recently. Herein we reported selective oxidation of furfural (FAL) to furoic acid (FA) with quantitative yield using whole-cells of Pseudomonas putida KT2440. The biocatalytic capacity could be substantially promoted through adding 5-hydroxymethylfurfural into media at the middle exponential growth phase. The reaction pH and cell dosage had notable impacts on both FA titer and selectivity. Based on the validation of key factors for FAL conversion, the capacity of P. putida KT2440 to produce FAL was substantially improved. In batch bioconversion, 170 mM FA was produced with selectivity nearly 100% in 2 h, whereas 204 mM FA was produced with selectivity above 97% in 3 h in fed-batch bioconversion. Particularly, the role of molybdate transporter in oxidation of FAL and 5-hydroxymethylfurfural was demonstrated for the first time. The furancarboxylic acids synthesis was repressed markedly by destroying molybdate transporter, which implied Mo-dependent enzyme/molybdoenzyme played pivotal role in such oxidation reactions. This research further highlights the potential of P. putida KT2440 as next generation industrial workhorse and provides a novel understanding of molybdoenzyme in oxidation of furanic aldehydes.

Transcriptome and metabolome analysis of Pichia stipitis to three representative lignocellulosic inhibitors.

During the bioconversion of xylose to ethanol, Pichia stipitis cells are often inhibited by substances generated in the lignocellulosic hydrolysate. However, the response mechanism of P. stipitis to inhibitors has not been completely understood till date. With this aim, integrated transcriptomic and metabolomic analyses were performed on P. stipitis to investigate the interactive effects of three representative inhibitors [vanillin, 5-hydroxymethylfurfural (5-HMF), and acetic acid] present in lignocellulosic hydrolysates. The genes involved in carbohydrate metabolism were observed to significantly down-regulated in the presence of the three combined inhibitors in both lag and middle exponential phases. In addition, inhibitor addition induced amino acid metabolism (e.g., glutamine and asparagine syntheses), since the yeast cells required more amino acids in stressful conditions. The metabolomic analysis yielded similar results, particularly those related with the analysis of metabolic biomarkers including fatty acids, amino acids, and sugars. 70 intracellular metabolites were detected by gas chromatography coupled with mass spectrometry (GC-MS), and samples from different phases were clearly separated by principal component analysis (PCA). The large amount of specific responsive genes and metabolites highlighted the complex regulatory mechanisms involved in the fermentation process in the presence of the three combined inhibitors.

Quantitative proteomic analysis of xylose fermentation strain Pichia stipitis CBS 5776 to lignocellulosic inhibitors acetic acid, vanillin and 5-hydroxymethylfurfural.

To obtain a global insight into the dynamic protein expression pattern in Pichia stipitis during xylose fermentation in the presence of three representative inhibitors (acetic acid, vanillin and 5-hydroxymethylfurfural), proteins were extracted for quantitative proteomic analysis using 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) on a liquid chromatography-mass/mass spectrometry instrument. Interestingly, aconitase (Aco1p) and NAD-isocitrate dehydrogenase (Idh1p) were upregulated during the middle exponential phase in the presence of the three inhibitors during tricarboxylic acid cycle. We speculated that yeast cells adaptively increased the expression of the tricarboxylic acid cycle proteins to compensate for low NADH derived from glycolysis in the presence of the three inhibitors. Proteins related to amino acid metabolism, aminoacyl tRNA synthesis and stress response were also significantly affected in the presence of the three inhibitors. Taken together, quantitative proteomic analysis is capable of monitoring P. stipitis xylose fermentation under inhibitor conditions and identifying physiological changes, such as stress response.

Characterization and pyrolysis behavior of the green microalga Micractinium conductrix grown in lab-scale tubular photobioreactor using Py-GC/MS and TGA/MS

Nowadays, microalgae are widely discussed as a promising feedstock for biofuel production. For higher crude bio-oil yield with good quality, microalgal biomass productivity and bio-oil characteristics are essential parameters. However, the same microalgal species has different chemical compositions at different growth phases. Therefore, the present study aimed to identify the best growth phase for high biomass productivity and optimal bio-oil production from the green microalga Micractinium conductrix via Py-GC/MS and TGA/MS analysis. M. conductrix was grown in a tubular photobioreactor and harvested at early exponential phase (EEP), middle exponential phase (MEP), late exponential phase (LEP) and stationary phase (STP). LEP showed the maximum significant (P ≤ 0.05) biomass productivity of 0.058 ± 0.004 g L−1 d-1, with maximum significant lipid and carbohydrate contents (28.7 ± 1.1 and 42.9 ± 1.2%dw, respectively). TGA/MS results confirmed that biomass harvested at MEP and LEP showed higher extent of conversion or mass loss reaction via thermal degradation with the lowest residual solid products. In addition, the hydrocarbon fragments in gaseous products (H2, C2H6, CH4, C2H4) from TGA/MS analysis were found to be released more abundantly at LEP. Moreover, Py-GC/MS results revealed that thermal decomposition of biomass harvested at LEP resulted in the highest significant relative contents of aliphatic hydrocarbons (41.2%) with lowest nitrogen-containing compounds (6.3%). The present study showed the significant impact of harvest time of microalgae on products characteristics of thermal decomposition and nominated LEP as the optimum growth phase to harvest M. conductrix for upgraded bio-oil production.

Growth and adherence of Staphylococcus aureus were enhanced through the PGE2 produced by the activated COX-2/PGE2 pathway of infected oral epithelial cells.

Staphylococcus aureus is a major pathogen of varieties of oral mucous infection. Prostaglandin E2 (PGE2) is a pro-inflammatory factor and Cyclooxygenase 2 (COX-2) is a critical enzyme of PGE2 biosynthesis. The purpose of this study is to investigate whether Staphylococcus aureus can increase PGE2 production of oral epithelial cells and how PGE2 functions in the growth and adherence of Staphylococcus aureus. mRNA levels of COX-2, fnbpA and fnbpB were estimated by quantitative PCR. PGE2 production was measured by Enzyme Linked Immunosorbent Assay (ELISA). The binding biomass of Staphylococcus aureus to human fibronectin was investigated by crystal violet staining and confocal laser scanning microscopy and the adherent force was measured by atomic force microscope (AFM). The COX-2 mRNA level and PGE2 production were increased by Staphylococcus aureus. PGE2 promoted the growth and biofilm formation of Staphylococcus aureus, enhanced the attachment of Staphylococcus aureus to the human fibronectin as well as to the HOK cells. The transcription of fnbpB was up-regulated by PGE2 in both early and middle exponential phase but not fnbpA. These results suggest that the activation of COX-2/PGE2 pathway in oral epithelial cell by Staphylococcus aureus can in turn facilitate the growth and the ability to adhere of the pathogen. These findings uncover a new function of PGE2 and may lead to the potential of COX-2/PGE2 targeting in the therapy of inflammation and cancer in both which the COX-2/PGE2 pathway were observed activated.

Production of selenium nanoparticles in Pseudomonas putida KT2440

Selenium (Se) is an essential element for the cell that has multiple applications in medicine and technology; microorganisms play an important role in Se transformations in the environment. Here we report the previously unidentified ability of the soil bacterium Pseudomonas putida KT2440 to synthesize nanoparticles of elemental selenium (nano-Se) from selenite. Our results show that P. putida is able to reduce selenite aerobically, but not selenate, to nano-Se. Kinetic analysis indicates that, in LB medium supplemented with selenite (1 mM), reduction to nano-Se occurs at a rate of 0.444 mmol L−1 h−1 beginning in the middle-exponential phase and with a final conversion yield of 89%. Measurements with a transmission electron microscope (TEM) show that nano-Se particles synthesized by P. putida have a size range of 100 to 500 nm and that they are located in the surrounding medium or bound to the cell membrane. Experiments involving dynamic light scattering (DLS) show that, in aqueous solution, recovered nano-Se particles have a size range of 70 to 360 nm. The rapid kinetics of conversion, easy retrieval of nano-Se and the metabolic versatility of P. putida offer the opportunity to use this model organism as a microbial factory for production of selenium nanoparticles.

Protective Role of Postbiotic Mediators Secreted by Lactobacillus rhamnosus GG Versus Lipopolysaccharide-induced Damage in Human Colonic Smooth Muscle Cells.

Some beneficial effects of probiotics may be due to secreted probiotic-derived factors, identified as "postbiotic" mediators. The aim of this study was to evaluate whether supernatants harvested from Lactobacillus rhamnosus GG (LGG) cultures (ATCC53103 strain) protect colonic human smooth muscle cells (HSMCs) from lipopolysaccharide (LPS)-induced myogenic damage. LGG was grown in de Man, Rogosa, Share medium at 37°C and samples were collected in middle and late exponential, stationary, and overnight phases. Supernatants were recovered by centrifugation, filtered, and stored at -20°C. The primary HSMCs culture was exposed for 24 hours to purified LPS of a pathogen strain of Escherichia coli (O111:B4) (1 μg/mL) with and without supernatants. Postbiotic effects were evaluated on the basis of HSMCs morphofunctional alterations and interleukin-6 (IL-6) production. Data are expressed as mean±SE (P<0.05 significant). LPS induced persistent, significant, 20.5%±0.7% cell shortening and 34.5%±2.2% decrease in acetylcholine-induced contraction of human HSMCs. These morphofunctional alterations were paralleled to a 365.65%±203.13% increase in IL-6 production. All these effects were dose-dependently reduced by LGG supernatants. Supernatants of the middle exponential phase already partially restored LPS-induced cell shortening by 57.34%±12.7% and IL-6 increase by 145.8%±4.3% but had no effect on LPS-induced inhibition of contraction. Maximal protective effects were obtained with supernatants of the late stationary phase with LPS-induced cell shortening restored by 84.1%±4.7%, inhibition of contraction by 85.5%±6.4%, and IL-6 basal production by 92.7%±1.2%. LGG-derived products are able to protect human SMCs from LPS-induced myogenic damage. Novel insights have been provided for the possibility that LGG-derived products could reduce the risk of progression to postinfective motor disorders.

Probing the Kinetic Anabolism of Poly-Beta-Hydroxybutyrate in Cupriavidus necator H16 Using Single-Cell Raman Spectroscopy.

Poly-beta-hydroxybutyrate (PHB) can be formed in large amounts in Cupriavidus necator and is important for the industrial production of biodegradable plastics. In this investigation, laser tweezers Raman spectroscopy (LTRS) was used to characterize dynamic changes in PHB content—as well as in the contents of other common biomolecule—in C. necator during batch growth at both the population and single-cell levels. PHB accumulation began in the early stages of bacterial growth, and the maximum PHB production rate occurred in the early and middle exponential phases. The active biosynthesis of DNA, RNA, and proteins occurred in the lag and early exponential phases, whereas the levels of these molecules decreased continuously during the remaining fermentation process until the minimum values were reached. The PHB content inside single cells was relatively homogenous in the middle stage of fermentation; during the late growth stage, the variation in PHB levels between cells increased. In addition, bacterial cells in various growth phases could be clearly discriminated when principle component analysis was performed on the spectral data. These results suggest that LTRS is a valuable single-cell analysis tool that can provide more comprehensive information about the physiological state of a growing microbial population.

Intracellular metabolic changes of Clostridium acetobutylicum and promotion to butanol tolerance during biobutanol fermentation

During the fermentation process, Clostridium acetobutylicum cells are often inhibited by the accumulated butanol. However, the mechanism underlying response of C. acetobutylicum to butanol stress remains poorly understood. This study was performed to clarify such mechanism through investigating the butanol stress-associated intracellular biochemical changes at acidogenesis phase (i.e., middle exponential phase) and solventogenesis phase (i.e., early stationary phase) by a gas chromatography-mass spectrometry-based metabolomics strategy. With the aid of partial least-squares-discriminant analysis, a pairwise discrimination between control group and butanol-treated groups was revealed, and 27 metabolites with variable importance in the projection value greater than 1 were identified. Under butanol stress, the glycolysis might be inhibited while TCA cycle might be promoted. Moreover, changes of lipids and fatty acids compositions, amino acid metabolism and osmoregulator concentrations might be the key factors involved in C. acetobutylicum metabolic response to butanol stress. It was suggested that C. acetobutylicum cells might change the levels of long acyl chain saturated fatty acids and branched-chain amino acids to maintain the integrity of cell membrane through adjusting membrane fluidity under butanol stress. The increased level of glycerol was considered to be correlated with osmoregulation and regulating redox balance. In addition, increased levels of some amino acids (i.e., threonine, glycine, alanine, phenylalanine, tyrosine, tryptophan, aspartate and glutamate) might also confer butanol tolerance to C. acetobutylicum. These results highlighted our knowledge about the response or adaptation of C. acetobutylicum to butanol stress, and would contribute to the construction of feasible butanologenic strains with higher butanol tolerance.

Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells.

Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent.

OP‐3 THE PROTECTIVE ROLE OF LACTOBACILLUS RHAMNOSUS GG‐DERIVED FACTORS AGAINST LPS‐INDUCED DAMAGE OF HUMAN COLONIC SMOOTH MUSCLE CELLS.

Impaired gut barrier function has been reported in some functional gastrointestinal (GI) disorders.Evidences suggest that gut microbiota affects GI motility in particular Lactobacillus species elicits anti‐inflammatory activity and exerts protective effects on damage induced by pathogen Gram negative‐derived lipopolysaccharide(LPS). LPS produced an oxidative imbalance in human colonic smooth muscle cells (SMC) that persists after LPS‐washout and contributes to SMC morphofunctional alterations. Aim: evaluate if supernatants harvested from LGG cultures protect SMC from LPS‐induced myogenic damage.Methods:L. rhamnosus GG (ATCC 53103 strain) was grown in MRS medium and samples were collected from bacterial cultures in middle exponential phase, in early, in middle and late stationary phase (overnight).Supernatants were recovered, filtered and stored at ‐20 °C. Highly pure human SMC culture was then exposed for 24 h to highly purified LPS (1 μg/ml) of E.coli (O111:B4) in the absence and presence of the supernatants.Their effects were evaluated on LPS‐induced SMC morphofunctional alterations and pro‐inflammatory IL‐6 production. Data are expressed as mean ± SE (p &lt; 0.05 significant).Results:LPS induced persistent significant 20.7% ± 1.2 cell shortening and 35.2% ± 2.6 decrease in contraction of human colonic SMC. These alterations were paralleled to a 238.5% ± 82.5 increase in IL‐6 production.These effects disappeared in the presence of LGG‐supernatants, following a progression related to LGG growth curve phases. Supernatants collected in the middle exponential phase already significantly partially restored LPS‐induced cell shortening by 43.4% ± 10.2 and IL6 increase by 47.6% ± 13.1 but had no effect on LPS‐induced inhibition of contraction. Supernatants collected later, in the early and middle stationary phase, further counteract LPS‐induced damage, including inhibition of contraction. Maximal protective effects were observed with supernatants of the late stationary phase where LPS‐induced cell shortening was reversed by 86% ± 4.7, inhibition of contraction by 98.2% ± 1.8 and IL6 basal production by 91.3% ± 0.6.Conclusions: LGG secreted products are substances/byproducts able to directly protect human SMC from LPS‐induced myogenic damage. Novel insights are then provided about the possibility that LGG‐derived products could reduce the risk of progression to a post‐infective motor disorder.

Production and properties of an exopolysaccharide synthesized by the extreme halophilic archaeon Haloterrigena turkmenica.

We have isolated a novel exopolysaccharide (EPS) produced by the extreme halophilic archaeon Haloterrigena turkmenica. Some features, remarkable from an industrial point of view, such as emulsifying and antioxidant properties, were investigated. H. turkmenica excreted 20.68 mg of EPS per 100 ml of culture medium when grown in usual medium supplemented with glucose. The microorganism excreted the biopolymer mainly in the middle exponential growth phase and reached the maximal production in the stationary phase. Analyses by anion exchange chromatography and SEC-TDA Viscotek indicated that the EPS was composed of two main fractions of 801.7 and 206.0 kDa. It was a sulfated heteropolysaccharide containing glucose, galactose, glucosamine, galactosamine, and glucuronic acid. Studies performed utilizing the mixture of EPS anionic fractions showed that the biopolymer had emulsifying activity towards vegetable oils comparable or superior to that exhibited by the controls, moderate antioxidant power when tested with 2,2'-diphenyl-1-picrylhydrazyl (DPPH(·)), and moisture-retention ability higher than hyaluronic acid (HA). The EPS from H. turkmenica is the first exopolysaccharide produced by an archaea to be characterized in terms of properties that can have potential biotechnological applications.

Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161.

This work aimed at characterizing the biochemical and biophysical properties of the membrane of Lactococcus lactis TOMSC161 cells during fermentation at different temperatures, in relation to their freeze-drying and storage resistance. Cells were cultivated at two different temperatures (22 and 30°C) and were harvested at different growth phases (from the middle exponential phase to the late stationary phase). Bacterial membranes were characterized by determining the fatty acid composition, the lipid phase transition, and the membrane fluidity. Cultivability and acidification activity losses of L. lactis were quantified after freezing, drying, and 3months of storage. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to lipid composition, and it was established that the cyclopropanation of unsaturated fatty acids with concomitant membrane rigidification during growth led to an increase in the freeze-drying and storage resistance of L. lactis. As expected, cultivating cells at a lower fermentation temperature than the optimum growth temperature induced a homeoviscous adaptation that was demonstrated by a lowered lipid phase transition temperature but that was not related to any improvement in freeze-drying resistance. L. lactis TOMSC161 was therefore able to develop a combined biochemical and biophysical response at the membrane level during fermentation. The ratio of cyclic fatty acids to unsaturated fatty acids (CFA/UFA) appeared to be the most relevant parameter associated with membrane rigidification and cell resistance to freeze-drying and storage. This study increased our knowledge about the physiological mechanisms that explain the resistance of lactic acid bacteria (LAB) to freeze-drying and storage stresses and demonstrated the relevance of complementary methods of membrane characterization.