Early Log Phase Research Articles

Novel Coating of Surgical Suture Confers Antimicrobial Activity Against Porphyromonas gingivalis and Enterococcus faecalis.

The oral cavity is colonized by >10(9) bacteria, many of which can increase heart disease risk when seeded into the bloodstream. Most dentoalveolar surgeries require the use of surgical sutures. Suture placement and removal can increase the risk of postoperative infection and bacteremia. The aim of this study is to evaluate the antimicrobial activity of a novel quaternary ammonium compound, K21, when coated on different suture materials. The periodontal pathogen Porphyromonas gingivalis and the endodontic species Enterococcus faecalis were grown to early log phase and inoculated on enriched Brucella blood agar, on which were placed identical lengths of surgical suture (chromic gut, polyester suture, silk, and nylon suture) and control unwaxed dental floss impregnated with K21 at 5%, 10%, 20%, and 25% volume/volume in ethanol vehicle. Controls included the following: 1) sutures treated with vehicle; 2) untreated sutures; and 3) unwaxed floss. Zones of inhibition in millimeters were measured at five randomized sites per suture/floss for each concentration and material used. Mean ± SD of zones of inhibition were calculated, and analysis of variance (P <0.05) was used to determine whether differences were statistically significant. The results indicate that K21-coated suture at concentrations ranging from 5% to 25%, depending on the type of suture, have antimicrobial activity for P. gingivalis and E. faecalis. Nylon suture coated with K21 at 5%, 10%, 20%, and 25% resulted in zones ranging from 3 to 11 mm. Polyester suture was more effective at lower K21 concentrations with 5% (P = 0.0031), 10% (P = 0.0011), and 20% (P = 0.0002), yielding 7.5, 8.3, and 10.5 mm zones of inhibition. K21-coated silk suture yielded significant zones of inhibition at 25% (P <0.0001), whereas chromic gut was effective at K21 concentrations of 5% (P = 0.0081) and 25% (P <0.0001). It is concluded that K21-coated surgical sutures have antimicrobial activity for bacterial species of direct relevance to postoperative infection and bacteremia.

Prokaryotic expression and purification of soluble goldfish Tgf2 transposase with transposition activity.

Goldfish Tgf2 transposon of Hobo/Activator/Tam3 (hAT) family can mediate gene insertion in a variety of aquacultural fish species by transposition; however, the protein structure of Tgf2 transposase (TPase) is still poorly understood. To express the goldfish Tgf2 TPase in Escherichia coli, the 2061-bp coding region was cloned into pET-28a(+) expression vector containing an N-terminal (His)6-tag. The pET-28a(+)-Tgf2 TPase expression cassette was transformed into Rosetta 1 (DE3) E. coli lines. A high yield of soluble proteins with molecular weight of ~80 kDa was obtained by optimized cultures including low-temperature (22 °C) incubation and early log phase (OD600 = 0.3-0.4) induction. Mass spectrometry analysis following trypsin digestion of the recombinant proteins confirmed a Tgf2 TPase component in the eluate of Ni(2+)-affinity chromatography. When co-injected into 1-2 cell embryos with a donor plasmid harboring a Tgf2 cis-element, the prokaryotic expressed Tgf2 TPase can mediate high rates (45 %) of transposition in blunt snout bream (Megalobrama amblycephala). Transposition was proved by the presence of 8-bp random direct repeats at the target sites, which is the signature of hAT family transposons. Production of the Tgf2 Tpase protein in a soluble and active form not only allows further investigation of its structure, but provides an alternative tool for fish transgenesis and insertional mutagenesis.

Evaluation of bactericidal activity of Hannon honey on slowly growing bacteria in the chemostat.

There is renewed interest in the therapeutic use of honey, including use in the treatment of infected wounds and burn patients. In this study, we have assessed the antibacterial activity of Libyan floral Hannon honey on Escherichia coli and Staphylococcus aureus, both known to infect wounds. The effects of four concentrations (5%–30%) of honey were compared with that of four antibiotics (ampicillin, tetracycline, polymyxin, and ciprofloxacin) on the growth of these bacteria at early log, mid log, and late log phases. It has been shown that E. coli and S. aureus are to some degree susceptible during mid log phase compared with late log phase, demonstrated by their complete resistance to antibiotics. Chemostat culture was used to investigate the effect of honey on E. coli grown at a steady state with specific growth rates between 0.1 to 0.5 hour−1. The rate of killing was distinctively clear during the two stages of growth monitored: there was a relatively moderate reduction at the slow growth phase (0.1 to 0.3 hour−1), while a dramatic reduction was obtained at the fast growth phase (0.3 to 0.5 hour−1), reaching a complete reduction at 0.5 hour−1. These results complement data using the cup-cut technique. The antibacterial effect of honey was concentration and time dependent, the bactericidal effect was indeed observed at low concentrations, it demonstrates that the honey has more impact on slow growing bacteria than antibiotics have. We suggest that more reduction could be achieved at higher concentrations of honey. These results may have important clinical implications, such as for the management of wound and burn patients.

Hierarchical Functional Specificity of Cytosolic Heat Shock Protein 70 (Hsp70) Nucleotide Exchange Factors in Yeast

Heat shock protein 70 (Hsp70) molecular chaperones play critical roles in protein homeostasis. In the budding yeast Saccharomyces cerevisiae, cytosolic Hsp70 interacts with up to three types of nucleotide exchange factors (NEFs) homologous to human counterparts: Sse1/Sse2 (Heat shock protein 110 (Hsp110)), Fes1 (HspBP1), and Snl1 (Bag-1). All three NEFs stimulate ADP release; however, it is unclear why multiple distinct families have been maintained throughout eukaryotic evolution. In this study we investigate NEF roles in Hsp70 cell biology using an isogenic combinatorial collection of NEF deletion mutants. Utilizing well characterized model substrates, we find that Sse1 participates in most Hsp70-mediated processes and is of particular importance in protein biogenesis and degradation, whereas Fes1 contributes to a minimal extent. Surprisingly, disaggregation and resolubilization of thermally denatured firefly luciferase occurred independently of NEF activity. Simultaneous deletion of SSE1 and FES1 resulted in constitutive activation of heat shock protein expression mediated by the transcription factor Hsf1, suggesting that these two factors are important for modulating stress response. Fes1 was found to interact in vivo preferentially with the Ssa family of cytosolic Hsp70 and not the co-translational Ssb homolog, consistent with the lack of cold sensitivity and protein biogenesis phenotypes for fes1Δ cells. No significant consequence could be attributed to deletion of the minor Hsp110 SSE2 or the Bag homolog SNL1. Together, these lines of investigation provide a comparative analysis of NEF function in yeast that implies Hsp110 is the principal NEF for cytosolic Hsp70, making it an ideal candidate for therapeutic intervention in human protein folding disorders.

Single-step fermentation of agricultural hemp residues for hydrogen and ethanol production

We have investigated a one-step fermentation process using purified hemp cellulose (PHC), hemp fibres (HF), and hemp hurds (HH), compared to reagent-grade α-cellullose (AC) for ethanol and hydrogen production by Clostridium thermocellum. Exponential phase growth and production rates on PHC were comparable to those observed with AC. Net production of ethanol for AC (8.47 mM) was only slightly higher than PHC (6.56 mM), but significantly higher than HF (5.48 mM) and HH (3.52 mM), while the final hydrogen yield was comparable for AC (12.70 mM), PHC (11.01 mM), HF (10.91 mM), and HH (4.72 mM). End-product yields were dependent on the intrinsic cellulosic content as well as the presence of other polymers present in substrate biomass. Rates of ethanol and hydrogen production were similar in early log phase but varied in mid-log, and accessibility to cellulose was shown to determine yield and metabolic flux distribution. Our data suggest that improving the accessibility of the cellulolytic bacterium, C. thermocellum, to cellulose fibres has a greater effect on increasing the yield of fermentation products than simply increasing the concentration of cellulose in the substrate.

English

Repeated use of L-asparaginase II enzyme, in the treatment of acute lymphoblastic leukaemia, is commonly needed because of the enzyme&rsquo;s instability and relatively short half-life which leads to more serious side effects on patients. In the present study, we report on the cloning and expression of L-asparaginase from a thermotolerant strain of Escherichia coli (KH027) which was isolated from camel manure and could grow at 45&deg;C. Expression of recombinant asparaginase was conducted by fusion asparaginase gene to pelB leader sequence and 6His residues at the C-terminus under the inducible T7 promoter in DH5&alpha; cells. Induction of the cells with 0.1 mM isopropyl-&beta;-D-thiogalactopyranoside (IPTG) at late log phase of growth resulted in 0.6-fold (2111 UI) higher to that obtained in early log phase induction (1319 UI) and 0.3-fold compared with mid log phase induction (1623 UI). The recombinant asparaginase protein was purified from the culture supernatant through nickel affinity chromatography. The apparent molecular weight of the tetramer enzyme was found to be ~141 kDa. Overall yield (87 mg/L) of the purified recombinant asparaginase was achieved at the shake flask level. The purified protein showed optimum activities at a temperature of 43&deg;C and pH 6. The Km and Kcat parameters were 3.8 mM-1 and 2.92 &times; 103s&minus;1, respectively. The enzyme retained around 57 and 30% of its initial activity after 30 and 60 min of incubation at 50&deg;C, respectively. Recombinant L-asparaginase was evaluated for its antiproliferative effect in the leukemia cell lines of RS4; 11 and HL60 after 96 and 72 h of incubation. The doses of 100 &mu;g/mL and time-response effect of 96 h caused a reduction value of 50% in cell viability of RS4. However, cell viability of 50% in the leukemic cells HL-60 was noticed with a concentration of 200 &mu;g/mL with an incubation period of 72 h. In vitro antiproliferative results in the leukemia cell lines encourage for making in vivo investigation to increase the possibility of using this thermostable enzyme in leukaemia therapy. &nbsp; Key words: L-Asparaginase II, leukemia cell, recombinant asparaginase.

Comparative genomics of the response to cold shock in Saccharomyces paradoxus and Saccharomyces cerevisiae (589.3)

The growth rate of a wild species of yeast, Saccharomyces paradoxus, was compared to the domesticated species, Saccharomyces cerevisiae, when both were subjected to different temperatures and inhibitory drugs. We found that S. paradoxus had a shorter doubling time than S. cerevisiae at both 30 and 13°C, and that S. paradoxus grew significantly faster at the cold temperature than was expected by its warm temperature growth rate. Furthermore, S. paradoxus was more resistant to the growth‐inhibitory drugs, rapamycin and caffeine, than S. cerevisiae. To determine the gene expression changes responsible for resistance to cold shock, we performed a DNA microarray experiment on S. paradoxus. Cells were grown to early log phase at 30°C, then shifted to 13°C for 60 minutes, and then shifted back to 30°C for another 60 minutes. Samples were collected before cold shock (t0), after 15, 30, and 60 minutes of cold shock (t15, t30, t60), and after 30 and 60 minutes (t90, t120) of recovery at 30°C. Labeled aRNA from cold shock and recovery timepoints were paired with labeled aRNA from the t0 timepoint. Four independent replicates of the experiment were performed with dye swaps for two of the replicates. Cluster analysis shows two dominant patterns of expression: up‐regulation during cold shock followed by down‐regulation during recovery and vice‐versa. Genes involved in ribosome biogenesis show the former pattern of expression, similar to the pattern observed in S. cerevisiae. Further analysis is being performed to discern differences in expression between the two species.Grant Funding Source: Supported by NSF RUI 0921038

Inhibition of the sodium-translocating NADH-ubiquinone oxidoreductase [Na+-NQR] decreases cholera toxin production in Vibrio cholerae O1 at the late exponential growth phase

Two virulence factors produced by Vibrio cholerae, cholera toxin (CT) and toxin-corregulated pilus (TCP), are indispensable for cholera infection. ToxT is the central regulatory protein involved in activation of CT and TCP expression. We previously reported that lack of a respiration-linked sodium-translocating NADH–ubiquinone oxidoreductase (Na+-NQR) significantly increases toxT transcription. In this study, we further characterized this link and found that Na+-NQR affects toxT expression only at the early-log growth phase, whereas lack of Na+-NQR decreases CT production after the mid-log growth phase. Such decreased CT production was independent of toxT and ctxB transcription. Supplementing a respiratory substrate, l-lactate, into the growth media restored CT production in the nqrA-F mutant, suggesting that decreased CT production in the Na+-NQR mutant is dependent on electron transport chain (ETC) activity. This notion was supported by the observations that two chemical inhibitors, a Na+-NQR specific inhibitor 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO) and a succinate dehydrogenase (SDH) inhibitor, thenoyltrifluoroacetone (TTFA), strongly inhibited CT production in both classical and El Tor biotype strains of V. cholerae. Accordingly, we propose the main respiratory enzyme of V. cholerae, as a potential drug target to treat cholera because human mitochondria do not contain Na+-NQR orthologs.

Transcriptome Changes Associated with Anaerobic Growth in Yersinia intermedia (ATCC29909)

BackgroundThe yersiniae (Enterobacteriaceae) occupy a variety of niches, including some in human and flea hosts. Metabolic adaptations of the yersiniae, which contribute to their success in these specialized environments, remain largely unknown. We report results of an investigation of the transcriptome under aerobic and anaerobic conditions for Y. intermedia, a non-pathogenic member of the genus that has been used as a research surrogate for Y. pestis. Y. intermedia shares characteristics of pathogenic yersiniae, but is not known to cause disease in humans. Oxygen restriction is an important environmental stimulus experienced by many bacteria during their life-cycles and greatly influences their survival in specific environments. How oxygen availability affects physiology in the yersiniae is of importance in their life cycles but has not been extensively characterized.Methodology/Principal FindingsTiled oligonucleotide arrays based on a draft genome sequence of Y. intermedia were used in transcript profiling experiments to identify genes that change expression in response to oxygen availability during growth in minimal media with glucose. The expression of more than 400 genes, constituting about 10% of the genome, was significantly altered due to oxygen-limitation in early log phase under these conditions. Broad functional categorization indicated that, in addition to genes involved in central metabolism, genes involved in adaptation to stress and genes likely involved with host interactions were affected by oxygen-availability. Notable among these, were genes encoding functions for motility, chemotaxis and biosynthesis of cobalamin, which were up-regulated and those for iron/heme utilization, methionine metabolism and urease, which were down-regulated.Conclusions/SignificanceThis is the first transcriptome analysis of a non-pathogenic Yersinia spp. and one of few elucidating the global response to oxygen limitation for any of the yersiniae. Thus this study lays the foundation for further experimental characterization of oxygen-responsive genes and pathways in this ecologically diverse genus.

A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon

Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log-phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.

Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature

BackgroundProducing recombinant plant proteins expressed in Escherichia coli produce in high yields and in a soluble and functional form can be difficult. Under overexpression conditions, proteins frequently accumulate as insoluble aggregates (inclusion bodies) within the producing bacteria. We evaluated how the initial culture density, temperature and duration of the expression stage affect the production of some eukaryotic enzymes in E. coli.FindingsA high yield of active soluble proteins was obtained by combining early-log phase cultures and low temperatures for protein induction. When IPTG was added at OD600 = 0.1 and cultures were maintained at 4°C for 48-72 h, the soluble protein yield was 3 fold higher than that obtained in the mid-log phase (OD600 = 0.6). Besides, the target protein expression increased and the endogenous bacterial proteins reduced, thus making the protein purification process easier and more efficient.ConclusionsThe protocol can be widely applied to proteins with a heterologous expression which was limited by loss of activity at high temperatures or by low soluble recombinant protein yield.

Triggers of Aggregation and Extracellular Polysaccharide Polymer Production in Acidovorax temperans

Bacterial aggregation has important implications for the maintenance of bacteria in engineered environments. The triggers for aggregation, however, are poorly understood. A strain of Acidovorax temperans CB2Hn isolated from activated sludge was found to exhibit transient aggregation and was applied as a model to investigate factors that regulate biological aggregation. Growth kinetic studies indicate CB2Hn has exponential growth rates (μ(max)) ranging from 0.11 to 0.75 (log(CFU mL(-1))h(-1)) depending on nutrient conditions. CB2Hn exhibited variable aggregation in growth media that differed in the type of available carbon. Aggregation indices and extracellular polysaccharide polymer levels showed transient maxima which occurred at different points in the growth curve for each medium type. Maximum aggregation points were detected at the beginning of log phase in media containing complex carbon sources. In contrast, maximum values were detected in early log phase and mid-to-late log phase in media containing both simple and complex carbon sources. The results suggest that aggregation is regulated by nutritional cues and is possibly triggered by the switch to utilisation of complex carbon substrates.

Tetracycline accelerates the temporally-regulated invasion response in specific isolates of multidrug-resistant Salmonella enterica serovar Typhimurium

BackgroundMultidrug-resistant (MDR) Salmonella isolates are associated with increased morbidity compared to antibiotic-sensitive strains and are an important health and safety concern in both humans and animals. Salmonella enterica serovar Typhimurium is a prevalent cause of foodborne disease, and a considerable number of S. Typhimurium isolates from humans and livestock are resistant to three or more antibiotics. The majority of these MDR S. Typhimurium isolates are resistant to tetracycline, a commonly used and clinically and agriculturally relevant antibiotic. Because exposure of drug-resistant bacteria to antibiotics can affect cellular processes associated with virulence, such as invasion, we investigated the effect tetracycline had on the invasiveness of tetracycline-resistant MDR S. Typhimurium isolates.ResultsThe isolates selected and tested were from two common definitive phage types of S. Typhimurium, DT104 and DT193, and were resistant to tetracycline and at least three other antibiotics. Although Salmonella invasiveness is temporally regulated and normally occurs during late-log growth phase, tetracycline exposure induced the full invasive phenotype in a cell culture assay during early-log growth in several DT193 isolates. No changes in invasiveness due to tetracycline exposure occurred in the DT104 isolates during early-log growth or in any of the isolates during late-log growth. Real-time PCR was used to test expression of the virulence genes hilA, prgH, and invF, and these genes were significantly up-regulated during early-log growth in most isolates due to tetracycline exposure; however, increased virulence gene expression did not always correspond with increased invasion, and therefore was not an accurate indicator of elevated invasiveness. This is the first report to assess DT193 isolates, as well as the early-log growth phase, in response to tetracycline exposure, and it was the combination of both parameters that was necessary to observe the induced invasion phenotype.ConclusionsIn this report, we demonstrate that the invasiveness of MDR S. Typhimurium can be modulated in the presence of tetracycline, and this effect is dependent on growth phase, antibiotic concentration, and strain background. Identifying the conditions necessary to establish an invasive phenotype is important to elucidate the underlying factors associated with increased virulence of MDR Salmonella.

Use of NAD(P)H Fluorescence Measurement for On-Line Monitoring of Metabolic State of Azohydromonas australica in Poly(3-hydroxybutyrate) Production

Culture fluorescence measurement is an indirect and non-invasive method of biomass estimation to assess the metabolic state of the microorganism in a fermentation process. In the present investigation, NAD(P)H fluorescence has been used for on-line in situ characterization of metabolic changes occurring during different phases of batch cultivation of Azohydromonas australica in growth associated poly(3-hydroxybutyrate) or PHB production. A linear correlation between biomass concentration and net NAD(P)H fluorescence was obtained during early log phase (3-12h) and late log phase (24-39h) of PHB fermentation. After 12h (mid log phase) cultivation PHB accumulation shot up and a drop in culture fluorescence was observed which synchronously exhibited continuous utilization of NAD(P)H for the synthesis of biomass and PHB formation simultaneously. A decrease in the observed net fluorescence value was observed again towards the end of fermentation (at 39h) which corresponded very well with the culture starvation and substrate depletion towards the end of cultivation inside the bioreactor. It was therefore concluded that NAD(P)H fluorescence measurements could be used for indication of the time of fresh nutrient (substrate) feed during substrate limitation to further enhance the PHB production.

Monitoring of population dynamics of Corynebacterium glutamicum by multiparameter flow cytometry

SummaryPhenotypic variation of microbial populations is a well-known phenomenon and may have significant impact on the success of industrial bioprocesses. Flow cytometry (FC) and the large repertoire of fluorescent dyes bring the high-throughput analysis of multiple parameters in single bacterial cells into reach. In this study, we evaluated a set of different fluorescent dyes for suitability in FC single cell analysis of the biotechnological platform organism Corynebacterium glutamicum. Already simple scattering properties of C. glutamicum cells in the flow cytometer were shown to provide valuable information on the growth activity of analysed cells. Furthermore, we used DAPI staining for a FC-based determination of the DNA content of C. glutamicum cells grown on standard minimal or complex media. Characteristic DNA patterns were observed mirroring the typical uncoupled DNA synthesis in the logarithmic (log) growth phase and are in agreement with a symmetric type of cell division of C. glutamicum. Application of the fluorescent dyes Syto 9, propidium iodide, and DiOC2(3) allowed the identification of subpopulations with reduced viability and membrane potential within early log and stationary phase populations. The presented data highlight the potential of FC-based analyses for online monitoring of C. glutamicum bioprocesses and provide a first reference for future applications and protocols.

Functional Analysis of Leishmania Cyclopropane Fatty Acid Synthetase

The single gene encoding cyclopropane fatty acid synthetase (CFAS) is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular) and host (intracellular) stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.

Surface tension and toxicity changes during biodegradation of carbazole by newly isolated methylotrophic strain Methylobacterium sp. GPE1

The novel Methylobacterium sp. GPE1 strain was isolated from a former gasworks site. GPE1 was able to utilise carbazole as a sole source of carbon and energy. Its ability to produce surface-active compounds was investigated by monitoring the biomass and surface tension. During the adaptation phase, the GPE1 cells cultured on carbazole (9.9 mg l−1) showed an unexpected change in the surface tension of the culture. The biodegradation process seemed to be initiated by the production of the biosurfactant. The appearance of an early log phase caused decrease of surface tension which reached its minimal value, equal to 61 mN m−1. The later surface tension changes were corresponding to biomass concentration and reached the control level at the end of experiment. In the 20 day experimental period, GPE1 utilised 87% of carbazole, giving the following values: μ = 0.08 h−1, Yxs = 0.62 gC gC−1. The Microtox™ toxicity assay was conducted for centrifuged and filtered water solutions of cultures and abiotic samples. The samples after biodegradation revealed the increased toxicity determined by luminescence inhibition which increased from 15.2% to 33%. Although the biosurfactant produced by GPE1 was not identified, its relation with signal molecules, which are often present in Methylobacterium sp. representatives, is being discussed.

Oxidative and Reductive Routes of Glycerol and Glucose Fermentation by Escherichia coli Batch Cultures and Their Regulation by Oxidizing and Reducing Reagents at Different pHs

Glycerol and glucose fermentation redox routes by Escherichia coli and their regulation by oxidizing and reducing reagents were investigated at different pHs. Cell growth was followed by decrease of pH and redox potential (E ( h )). During glycerol utilization at pH 7.5 ∆pH, the difference between initial and end pH, was lower compared with glucose fermentation. After 8 h growth, during glycerol utilization E ( h ) dropped down to negative values (-150 mV) but during glucose fermentation it was positive (+50 mV). In case of glycerol H(2) was evolved at the middle log phase while during glucose fermentation H(2) was produced during early log phase. Furthermore, upon glycerol utilization, oxidizer potassium ferricyanide (1 mM) inhibited both cell growth and H(2) formation. Reducing reagents DL-dithiothreitol (3 mM) and dithionite (1 mM) inhibited growth but stimulated H(2) production. The findings point out the importance of reductive conditions for glycerol fermentation and H(2) production by E. coli.

Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism

The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been examined to analyze the maximal rate of initial transformation, determine the effects of metabolic mutations of the host on transformation rate, and to assess the cell metabolic changes brought about during TNT transformation. Little difference in the maximal rate of TNT degradation in early acid phase cultures was found between the parental ATCC 824 strain and strains altered in the acid forming pathways (phosphotransacetylase, or butyrate kinase) or in a high-solvent-producing strain (mutant B). This result is in agreement with the previous findings of a similar degradation rate in a degenerate strain (M5) that had lost the ability to produce solvent. A series of antisense constructs were made that reduced the expression of hydA, encoding the Fe-hydrogenase, or hydE and hydF, genes encoding hydrogenase maturating proteins. While the antisense hydA strain had only ∼30 % of the activity of wild type, the antisense hydE strain exhibited a TNT degradation rate around 70 % that of the parent. Overexpression of hydA modestly increased the TNT degradation rate in acid phase cells, suggesting the amount of reductant flowing into hydrogenase rather than the hydrogenase level itself was a limiting factor in many situations. The redox potential, hydrogen evolution, and organic acid metabolites produced during rapid TNT transformation in early log phase cultures were measured. The redox potential of the acid-producing culture decreased from -370 to -200 mV immediately after addition of TNT and the hydrogen evolution rate decreased, lowering the hydrogen to carbon dioxide ratio from 1.4 to around 1.1 for 15 min. During the time of TNT transformation, the treated acidogenic cells produced less acetate and more butyrate. The results show that during TNT transformation, the cells shift metabolism away from hydrogen formation to reduction of TNT and the resulting effects on cell redox cofactors generate a higher proportion of butyrate.

Ksp1 Kinase Regulates Autophagy via the Target of Rapamycin Complex 1 (TORC1) Pathway

Macroautophagy (hereafter autophagy) is a bulk degradation system conserved in all eukaryotes, which engulfs cytoplasmic components within double-membrane vesicles to allow their delivery to, and subsequent degradation within, the vacuole/lysosome. Autophagy activity is tightly regulated in response to the nutritional state of the cell and also to maintain organelle homeostasis. In nutrient-rich conditions, Tor kinase complex 1 (TORC1) is activated to inhibit autophagy, whereas inactivation of this complex in response to stress leads to autophagy induction; however, it is unclear how the activity of TORC1 is controlled to allow precise adjustments in autophagy activity. In this study, we performed genetic analyses in Saccharomyces cerevisiae to identify factors that regulate TORC1 activity. We determined that the Ksp1 kinase functions in part as a negative regulator of autophagy; deletion of KSP1 facilitated dephosphorylation of Atg13, a TORC1 substrate, which correlates with enhanced autophagy. These results suggest that Ksp1 down-regulates autophagy activity via the TORC1 pathway. The suppressive function of Ksp1 is partially activated by the Ras/cAMP-dependent protein kinase A (PKA), which is another negative regulator of autophagy. Our study therefore identifies Ksp1 as a new component that functions as part of the PKA and TORC1 signaling network to control the magnitude of autophagy.