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Minimal Medium (minimal + medium)
Selected AbstractsCharacterizing the regulation of the Pu promoter in Acinetobacter baylyi ADP1ENVIRONMENTAL MICROBIOLOGY, Issue 7 2008Wei E. Huang Summary Effective gene trapping and screening requires sensory and regulatory compatibility of both host and exogenous systems. The naturally competent bacterium Acinetobacter baylyi ADP1 is able to efficiently take up and integrate exogenous DNA into the chromosome, making it an attractive host system for a wide range of metagenomic applications. To test the ability of A. baylyi ADP1 to express the XylR-regulated Pu promoter from Pseudomonas putida mt-2, we have constructed and examined an A. baylyi ADP1 strain, ADPWH- Pu-lux-xylR. The Pu promoter in ADPWH- Pu-lux-xylR was specifically induced by toluene, m -, p - and o- xylene. The substrate-induced Pu promoter was highly dependent on the growth medium: it was repressed in rich media until stationary phase, but was immediately induced in minimal medium with glucose as the sole carbon source (MMG). However, the Pu promoter was repressed in MMG when it was supplemented with 5 g l,1 yeast extract. Further investigation showed that the Pu promoter in MMG was repressed by 0.5 g l,1 aspartic acid or asparagine, but not repressed by glutamine. Changing the carbon/nitrogen ratios by addition of ammonia did not significantly affect the Pu promoter activity but addition of nitrate did. These results show that A. baylyi ADP1 reproduced characteristics of the XylR-regulated Pu promoter observed in its original host. It demonstrates that A. baylyi could provide an excellent genetic host for a wide range of functional metagenomic applications. [source] Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and functionENVIRONMENTAL MICROBIOLOGY, Issue 8 2007Wei E. Huang Summary We have coupled fluorescence in situ hybridization (FISH) with Raman microscopy for simultaneous cultivation-independent identification and determination of 13C incorporation into microbial cells. Highly resolved Raman confocal spectra were generated for individual cells which were grown in minimal medium where the ratio of 13C to 12C content of the sole carbon source was incrementally varied. Cells which were 13C-labelled through anabolic incorporation of the isotope exhibited key red-shifted spectral peaks, the calculated ,red shift ratio' (RSR) being highly correlated with the 13C-content of the cells. Subsequently, Raman instrumentation and FISH protocols were optimized to allow combined epifluorescence and Raman imaging of Fluos, Cy3 and Cy5-labelled microbial populations at the single cell level. Cellular 13C-content determinations exhibited good congruence between fresh cells and FISH hybridized cells indicating that spectral peaks, including phenylalanine resonance, which were used to determine 13C-labelling, were preserved during fixation and hybridization. In order to demonstrate the suitability of this technology for structure,function analyses in complex microbial communities, Raman-FISH was deployed to show the importance of Pseudomonas populations during naphthalene degradation in groundwater microcosms. Raman-FISH extends and complements current technologies such as FISH-microautoradiography and stable isotope probing in that it can be applied at the resolution of single cells in complex communities, is quantitative if suitable calibrations are performed, can be used with stable isotopes and has analysis times of typically 1 min per cell. [source] Fission yeast decaprenyl diphosphate synthase consists of Dps1 and the newly characterized Dlp1 protein in a novel heterotetrameric structureFEBS JOURNAL, Issue 20 2003Ryoichi Saiki The analysis of the structure and function of long chain-producing polyprenyl diphosphate synthase, which synthesizes the side chain of ubiquinone, has largely focused on the prokaryotic enzymes, and little is known about the eukaryotic counterparts. Here we show that decaprenyl diphosphate synthase from Schizosaccharomyces pombe is comprised of a novel protein named Dlp1 acting in partnership with Dps1. Dps1 is highly homologous to other prenyl diphosphate synthases but Dlp1 shares only weak homology with Dps1. We showed that the two proteins must be present simultaneously in Escherichia coli transformants before ubiquinone-10, which is produced by S. pombe but not by E. coli, is generated. Furthermore, the two proteins were shown to form a heterotetrameric complex. This is unlike the prokaryotic counterparts, which are homodimers. The deletion mutant of dlp1 lacked the enzymatic activity of decaprenyl diphosphate synthase, did not produce ubiquinone-10 and had the typical ubiquinone-deficient S. pombe phenotypes, namely hypersensitivity to hydrogen peroxide, the need for antioxidants for growth on minimal medium and an elevated production of H2S. Both the dps1 (formerly dps) and dlp1 mutants could generate ubiquinone when they were transformed with a bacterial decaprenyl diphosphate synthase, which functions in its host as a homodimer. This indicates that both dps1 and dlp1 are required for the S. pombe enzymatic activity. Thus, decaprenyl diphosphate from a eukaryotic origin has a heterotetrameric structure that is not found in prokaryotes. [source] Ycf1p-dependent Hg(II) detoxification in Saccharomyces cerevisiaeFEBS JOURNAL, Issue 11 2003Olivier Gueldry In Saccharomyces cerevisiae, disruption of the YCF1 gene increases the sensitivity of cell growth to mercury. Transformation of the resulting ycf1 null mutant with a plasmid harbouring YCF1 under the control of the GAL promoter largely restores the wild-type resistance to the metal ion. The protective effect of Ycf1p against the toxicity of mercury is especially pronounced when yeast cells are grown in rich medium or in minimal medium supplemented with glutathione. Secretory vesicles from S. cerevisiae cells overproducing Ycf1p are shown to exhibit ATP-dependent transport of bis(glutathionato)mercury. Moreover, using ,-galactosidase as a reporter protein, a relationship between mercury addition and the activity of the YCF1 promoter can be shown. Altogether, these observations indicate a defence mechanism involving an induction of the expression of Ycf1p and transport by this protein of mercury,glutathione adducts into the vacuole. Finally, possible coparticipation in mercury tolerance of other ABC proteins sharing close homology with Ycf1p was investigated. Gene disruption experiments enable us to conclude that neither Bpt1p, Yor1p, Ybt1p nor YHL035p plays a major role in the detoxification of mercury. [source] Bacterial competition between a bacteriocin-producing and a bacteriocin-negative strain of Streptococcus bovis in batch and continuous cultureFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Bruno M. Xavier Abstract A bacteriocin-producing Streptococcus bovis strain (HC5) outcompeted a sensitive strain (JB1) before it reached stationary phase (pH 6.4), even though it grew 10% slower and cell-free bovicin HC5 could not yet be detected. The success of bacteriocin-negative S. bovis isolates was enhanced by the presence of another sensitive bacterium (Clostridium sticklandii SR). PCR based on repetitive DNA sequences indicated that S. bovis HC5 was not simply transferring bacteriocin genes to S. bovis JB1. When the two S. bovis strains were coinoculated into minimal medium, bacteriocin-negative isolates predominated, and this effect could be explained by the longer lag time (0.5 vs. 1.5 h) of S. bovis HC5. If the glucose concentration of the minimal medium was increased from 2 to 7 mg mL,1, the effect of lag time was diminished and bacteriocin-producing isolates once again dominated the coculture. When the competition was examined in continuous culture, it became apparent that batch culture inocula were never able to displace a strain that had already reached steady state, even if the inoculum was large. This result indicated that bacterial selection for substrate affinity was even more important than bacteriocin production. [source] Glycolysis in Ustilago maydisFEMS YEAST RESEARCH, Issue 8 2008Emma Saavedra Abstract The kinetic parameters of the 10 glycolytic enzymes and glycolytic fluxes were determined for the first time in Ustilago maydis. Enzyme activities in yeast grown in minimal medium and harvested in the stationary stage were twofold higher than those from yeast grown in rich medium. In contrast, in yeast harvested in the exponential stage, the enzyme activities were higher in cells grown in rich medium. Phosphofructokinase activity was the lowest in the four culture conditions analyzed, suggesting that this enzyme is a flux-controlling step in U. maydis glycolysis. The Vmax and Km values of hexokinase and pyruvate kinase were similar under all conditions. The results revealed that U. maydis aldolase belongs to the class II type of metalo-aldolases. 3-Phosphoglycerate mutase (PGAM) activity was 2,3-bisphosphoglycerate cofactor independent, which contrasted with the cofactor dependency predicted by the amino acid sequence alignment analysis. Pyruvate was secreted by U. maydis yeast in the presence and absence of external glucose. The glycolytic enzyme activities in the U. maydis mycelial form were similar to those found in yeast, except for one order of magnitude higher phosphofructokinase and PGAM activities, thus suggesting differences in the glycolysis regulatory mechanisms between the two cellular forms. [source] Viable ultramicrocells in drinking waterJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009F.S. Silbaq Abstract Aims:, To examine the diversity of cultivable 0·2 micron filtrate biofilm forming bacteria from drinking water systems. Methods and Results:, Potable chlorinated drinking water hosts phylogenetically diverse ultramicrocells (UMC) (0·2 and 0·1 ,m filterable). UMC (starved or dwarf bacteria) were isolated by cultivation on minimal medium from a flow system wall model with polyvinyl chloride (PVC) pipes. All cultivated cells (25 different isolates) did not maintain their ultra-size after passages on rich media. Cultured UMC were identified by their 16S ribosomal DNA sequences. The results showed that they were closely related to uncultured and cultured members of the Proteobacteria, Actinobacteria and Firmicutes. The isolates of phylum Actinobacteria included representatives of a diverse set of Actinobacterial families: Micrococcaceae, Microbacteriaceae, Dermabacteraceae, Nocardiaceae and Nocardioidaceae. Conclusions:, This study is the first to show an abundance of cultivable UMC of various phyla in drinking water system, including a high frequency of bacteria known to be involved in opportunistic infections, such as Stenotrophomonas maltophilia, Microbacterium sp., Pandoraea sp. and Afipia strains. Significance and Impact of the Study:, Chlorinated tap water filtrate (0·2 and 0·1 ,m) still harbours opportunistic micro-organisms that can pose some health threat. [source] Ectoines as compatible solutes and carbon and energy sources for the halophilic bacterium Chromohalobacter salexigensJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2006C. Vargas Abstract Aims:, To investigate the catabolism of ectoine and hydroxyectoine, which are the major compatible solutes synthesized by Chromohalobacter salexigens. Methods and Results:, Growth curves performed in M63 minimal medium with low (0·75 mol l,1 NaCl), optimal (1·5 mol l,1 NaCl) or high (2·5 mol l,1 NaCl) salinity revealed that betaine and ectoines were used as substrate for growth at optimal and high salt. Ectoine transport was maximal at optimal salinity, and showed 3- and 1·5-fold lower values at low and high salinity respectively. The salt-sensitive ectA mutant CHR62 showed an ectoine transport rate 6·8-fold higher than that of the wild type. Incubation of C. salexigens in a mixture of glucose and ectoine resulted in a biphasic growth pattern. However, CO2 production due to ectoine catabolism was lower, but not completely abolished, in the presence of glucose. When used as the sole carbon source, glycine betaine effectively inhibited ectoine and hydroxyectoine synthesis at any salinity. Conclusions:, The catabolic pathways for ectoine and hydroxyectoine in C. salexigens operate at optimal and high (although less efficiently) salinity. Endogenous ectoine(s) may repress its own transport. Ectoine utilization was only partially repressed by glucose. Betaine, when used as carbon source, suppresses synthesis of ectoines even under high osmolarity conditions. Significance and Impact of the Study:, This study is a previous step to the subsequent isolation and manipulation of the catabolic genes, so as to generate strains with enhanced production of ectoine and hydroxyectoine. [source] Autophagy and amino acid homeostasis are required for chronological longevity in Saccharomyces cerevisiaeAGING CELL, Issue 4 2009Ashley L. Alvers Summary Following cessation of growth, yeast cells remain viable in a nondividing state for a period of time known as the chronological lifespan (CLS). Autophagy is a degradative process responsible for amino acid recycling in response to nitrogen starvation and amino acid limitation. We have investigated the role of autophagy during chronological aging of yeast grown in glucose minimal media containing different supplemental essential and nonessential amino acids. Deletion of ATG1 or ATG7, both of which are required for autophagy, reduced CLS, whereas deletion of ATG11, which is required for selective targeting of cellular components to the vacuole for degradation, did not reduce CLS. The nonessential amino acids isoleucine and valine, and the essential amino acid leucine, extended CLS in autophagy-deficient as well as autophagy-competent yeast. This extension was suppressed by constitutive expression of GCN4, which encodes a transcriptional regulator of general amino acid control (GAAC). Consistent with this, GCN4 expression was reduced by isoleucine and valine. Furthermore, elimination of the leucine requirement extended CLS and prevented the effects of constitutive expression of GCN4. Interestingly, deletion of LEU3, a GAAC target gene encoding a transcriptional regulator of branched side chain amino acid synthesis, dramatically increased CLS in the absence of amino acid supplements. In general, this indicates that activation of GAAC reduces CLS whereas suppression of GAAC extends CLS in minimal medium. These findings demonstrate important roles for autophagy and amino acid homeostasis in determining CLS in yeast. [source] Cooperative biodegradation of geosmin by a consortium comprising three gram-negative bacteria isolated from the biofilm of a sand filter columnLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2006D. Hoefel Abstract Aims:, To isolate and identify bacteria from a sand filter column capable of degrading the taste and odour compound, geosmin. In doing so, to investigate if these organisms degrade geosmin either individually or if an alternative mechanism is utilized. Methods and Results:, Geosmin-degrading bacteria from a biologically active sand filter column were enriched by their growth in a minimal medium supplemented with geosmin as the sole carbon source. By day 51, 21·7 mg l,1 of geosmin had been degraded as determined by solid-phase microextraction gas chromatography/mass spectrometry, and was accompanied by a 2·12 log10 increase in active bacterial numbers as measured using the BacLightTM bacterial viability kit and flow cytometric enumeration. During the onset of geosmin degradation, the predominance of three bacteria, most similar to previously cultured species of Sphingopyxis alaskensis, Novosphingobium stygiae and Pseudomonas veronii based on 16S rRNA gene sequences was detected by denaturing gradient gel electrophoresis. Subsequent isolation of these organisms revealed that degradation of geosmin, when present as either the sole carbon source (ranging from 40 ng l,1 to 20 mg l,1) or when spiked into sterile reservoir water (37 and 131 ng l,1), occurred only when all three isolates were present. None of the isolates was shown to be capable of degrading geosmin either individually or in any combination of two. Conclusions:, This study has reported, for the first time, the cooperative degradation of geosmin by a consortium comprising three gram-negative bacteria isolated from a biologically active sand filter column. Significance and Impact of the Study:, These results are important for researchers currently employing molecular-based approaches to further understand the biodegradation of geosmin by bacteria, as such studies may be complicated by the discovery of geosmin degradation occurring by a consortium. This study also advances the knowledge surrounding the types of bacteria capable of degrading the taste and odour compound, as investigations to date regarding this are limited. [source] Expression of an artificial polypeptide with a repeated tripeptide glutamyl,tryptophanyl,lysine in Saccharomyces cerevisiaeLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2003S.Y. Lee Abstract Aims: Artificial genes, which encode 48 or 64 repeats of a tripeptide, glutamyl,tryptophanyl,lysine have been cloned to the yeast expression vector pAM82 containing the PHO5 promoter and expressed in Saccharomyces cerevisiae AH22. Methods and Results: When the yeast cells harbouring recombinant plasmids pALTG6-2 and pALTG4-4 were derepressed in Burkholder minimal medium (Toh-e, A., Ueda, Y., Kakimoto, S.I. and Oshima, Y. (1973) Journal of Bacteriology113, 727,738) containing low phosphate (0·03 g l,1 KH2PO4 and 1·5 g l,1 KCl), the expression was the highest after 24 h induction and the artificial polypeptides were synthesized to about 10% (pALTG6-2) and 14% (pALTG4-4) of the total cell protein. Conclusions: The artificial polypeptides produced in yeast were made to react with the rabbit antiserum against the polypeptide purified from Escherichia coli and found only in the pellet fraction of cell lysates, indicating the formation of inclusion body. Artificial polypeptide consisting of Glu,Trp,Lys may be useful as partial supplement in food and feeds. Significance and Impact of the Study: The production of single cell enriched with homopolymers of an essential amino acid in yeast might be an important tool of supplementing cereal diets and feed grain rations and could be used as means for improvement of the amino acid profile of single cell protein and production of pharmaceutical peptides. [source] Rhizoremediation of lindane by root-colonizing SphingomonasMICROBIAL BIOTECHNOLOGY, Issue 1 2008Dietmar Böltner Summary We used a two-step enrichment approach to isolate root-colonizing hexachlorocyclohexane (HCH)-degrading microorganisms. The first step consists of the use of classical liquid enrichment to isolate ,-HCH degraders. The ,-HCH-degrading microbes were attached in mass to corn seeds sown in soil with ,-HCH, and after plant development we rescued bacteria growing on root tips. Bacteria were then subjected to a second enrichment round in which growth on liquid medium with ,-HCH and inoculation of corn seeds were repeated. We then isolated bacteria on M9 minimal medium with ,-HCH from root tips. We were able to isolate four Sphingomonas strains, all of which degraded ,-, ,-, ,- and ,-HCH. Two of the strains were particularly good colonizers of corn roots, reaching high cell density in vegetated soil and partly removing ,-HCH. In contrast, these bacteria performed poorly in unplanted soils. This study supports the hypothesis that the removal of persistent toxic chemicals can be accelerated by combinations of plants and bacteria, a process generally known as rhizoremediation. [source] Three gene products govern (p)ppGpp production by Streptococcus mutansMOLECULAR MICROBIOLOGY, Issue 6 2007José A. Lemos Summary The current dogma implicating RelA as the sole enzyme controlling (p)ppGpp production and degradation in Gram-positive bacteria does not apply to Streptococcus mutans. We have now identified and characterized two genes, designated as relP and relQ, encoding novel enzymes that are directly involved in (p)ppGpp synthesis. Additionally, relP is co-transcribed with a two-component signal transduction system (TCS). Analysis of the (p)ppGpp synthetic capacity of various mutants and the behaviour of strains lacking combinations of the synthetase enzymes have revealed a complex regulon and fundamental differences in the way S. mutans manages alarmone production compared with bacterial paradigms. The functionality of the RelP and RelQ enzymes was further confirmed by demonstrating that expression of relP and relQ restored growth of a (p)ppGpp0Escherichia coli strain in minimal medium, SMG and on medium containing 3-amino-1,2,4-triazole, and by demonstrating (p)ppGpp production in various complemented mutant strains of E. coli and S. mutans. Notably, RelQ, and RelP and the associated TCS, are harboured in some, but not all, pathogenic streptococci and related Gram-positive organisms, opening a new avenue to explore the variety of strategies employed by human and animal pathogens to survive in adverse conditions that are peculiar to environments in their hosts. [source] The rpf gene of Micrococcus luteus encodes an essential secreted growth factorMOLECULAR MICROBIOLOGY, Issue 3 2002Galina V. Mukamolova Summary Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the ,Rpf domain' is fully active. The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease. [source] Identification of novel hrp -regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genomeMOLECULAR MICROBIOLOGY, Issue 5 2002Julie Zwiesler-Vollick Summary Pseudomonas syringae pv. tomato ( Pst ) strain DC3000 infects the model plants Arabidopsis thaliana and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by Pst DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in Pst DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of P. syringae are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved cis element (called the ,hrp box') in the promoters of type III secretion-associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published hrp operons and putative effector genes in Pst DC3000. Seventy-three predicted genes were retrieved from the January 2001 release of the Pst DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in hrp -inducing minimal medium than in nutrient-rich Luria,Bertani broth. Expression of all eight genes was dependent on the hrpS gene. Most were also dependent on the hrpL gene, but at least one was dependent on the hrpS gene, but not on the hrpL gene. An AvrRpt2-based type III translocation assay provides evidence that some of the hrpS -regulated novel genes encode putative effector proteins. [source] Stress and the periodontal diseases: growth responses of periodontal bacteria to Escherichia coli stress-associated autoinducer and exogenous FeMOLECULAR ORAL MICROBIOLOGY, Issue 3 2005A. Roberts Psychological stress is known to increase the circulating levels of the catecholamine hormones noradrenaline and adrenaline, which have been shown to influence the growth of a large number of bacterial species by acting in a siderophore-like manner or by inducing the production of novel autoinducers of growth. As we have previously demonstrated that periodontal organisms display differing growth responses to noradrenaline and adrenaline, the aim of this study was to determine whether these growth effects were based upon either siderophore-like or autoinducer mechanisms. Initial inocula of 43 microbial organisms normally found within the subgingival biofilm were established under anaerobic conditions (35°C). Each strain was re-inoculated into a serum-based minimal medium and growth was assessed by optical density (OD600nm) with test and control cultures performed in triplicate. Test cultures were supplemented with either 50 ,m ferric nitrate or a previously described Escherichia coli autoinducer of growth. Significant growth effects for supplementation with ferric nitrate (13 species responding positively) and E. coli autoinducer (24 species responding positively) were observed, with differences in growth response within bacterial species and within microbial complexes. When data for all organisms were compared with published responses to catecholamines there were only weak correlations with Fe (r = 0.28) and E. coli autoinducer (r = 0.34) responses. However, large positive responses (> 25% increase) to free Fe and/or E. coli autoinducer were significantly more prevalent in the group of organisms (n = 12) known to exhibit similar responses to catecholamine hormones (P < 0.01; ,2 = 4.56). The results support the view that catecholamines may exert their effects on subgingival organisms by initiating autoinducer production, or simply by acting in a siderophore-like manner, scavenging bound iron from the local environment. It is possible that autoinducer mechanisms may play an important role in the response of oral microorganisms to stress hormones, thereby contributing to the clinical course of stress-associated periodontal diseases. [source] Hexose-specificity of hexokinase and ADP-dependence of pyruvate kinase play important roles in the control of monosaccharide utilization in freshly diluted boar spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2006Antonio Medrano Abstract Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L -lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7,±,0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3,±,0.1 mIU/mg protein to 0.60,±,1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly dependent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15,0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose transporter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins involved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at concrete points in the boar-sperm surface. Mol. Reprod. Dev. 1179,1194, 2006. © 2006 Wiley-Liss, Inc. [source] Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: The role of iron in cell growth and virulence factor inductionBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2010Beum Jun Kim Abstract The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086,h,1 with an initial cell mass of less than 0.6,OD600. Damped oscillations and negative growth rates are unusual observations for bacterial systems. We have observed these responses at values of initial cell mass of 0.9,OD600 or higher, or at low dilution rates (<0.05,h,1) irrespectively of initial cell mass. This response suggests complex dynamics including the possibility of multiple steady states. Iron, which was reported earlier as a growth limiting nutrient in a widely used minimal medium, enhances both growth and virulence factor induction in iron-supplemented cultures compared to unsupplemented controls. Intracellular iron concentration is correlated to the early induction (6,h) of virulence factors in both batch and chemostat cultures. A reduction in aconitase activity (a TCA cycle enzyme) and ATP levels in iron-limited chemostat cultures was observed compared to iron-supplemented chemostat cultures, indicating that iron affects central metabolic pathways. We conclude that DC3000 cultures are particularly dependent on the environment and iron is likely a key nutrient in determining physiology. Biotechnol. Bioeng. 2010;105: 955,964. © 2009 Wiley Periodicals, Inc. [source] Microbial bio-production of a recombinant stimuli-responsive biosurfactantBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009W. Kaar Abstract Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states. Biotechnol. Bioeng. 2009;102: 176,187. © 2008 Wiley Periodicals, Inc. [source] Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell cultureBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2008Aleksei Rozkov Abstract Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h,1) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68,107 g,L,1 wet weight) achieves high volumetric yields of plasmid (95,277 mg,L,1 depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg,1 or less. Biotechnol. Bioeng. 2008;99: 557,566. © 2007 Wiley Periodicals, Inc. [source] Development of a minimal defined medium for recombinant human interleukin-3 production by Streptomyces lividans 66BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2008Keyvan Nowruzi Abstract A systematic approach was developed to identify and optimize the essential amino acids in defined minimal medium for the production of recombinant human interleukin 3 (rHuIL-3) by Streptomyces lividans. Starvation trials were carried out initially to narrow down the number of probable essential amino acids from an initial number of 20 to 8. Then a screening mixture experiment was designed and performed with the eight identified amino acids and distance-based multivariate analysis was employed to rank the probable essential amino acids regarding both growth and product formation. Following this procedure, the search was narrowed to four amino acids (Asp, Leu, Met, and Phe). Finally, a mixture design experiment known as the simplex lattice design was carried out and the composition of the optimum minimal medium was found (Asp 53%, Met 5%, and Phe 42%). Biotechnol. Bioeng. 2008;99: 214,222. © 2007 Wiley Periodicals, Inc. [source] Invariability of central metabolic flux distribution in Shewanella oneidensis MR-1 under environmental or genetic perturbationsBIOTECHNOLOGY PROGRESS, Issue 5 2009Yinjie J. Tang Abstract An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time ,3 h), salt stressed minimal medium (doubling time ,6 h), and minimal medium with amino acid supplementation (doubling time ,1.5 h). 13C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, 10 transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] A Novel Genetic Selection System for Improved Enantioselectivity of Bacillus subtilis Lipase ACHEMBIOCHEM, Issue 7 2008Ykelien L. Boersma Dr. Abstract In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for. In this study, we present a successful strategy that not only selects for catalytic activity, but for the first time also for enantioselectivity, as demonstrated by the selection of Bacillus subtilis lipase A variants with inverted and improved enantioselectivity. A lipase mutant library in an aspartate auxotroph Escherichia coli was plated on minimal medium that was supplemented with the aspartate ester of the desired enantiomer (S)-(+)-1,2- O -isopropylidene- sn -glycerol. To inhibit growth of less enantioselective variants, a covalently binding phosphonate ester of the opposite (R)-(,)-1,2- O -isopropylidene- sn -glycerol enantiomer was added as well. After three selection rounds in which the selection pressure was increased by raising the phosphonate ester concentration, a mutant was selected with an improved enantioselectivity increased from an ee of ,29.6,% (conversion 23.4,%) to an ee of +73.1,% (conversion 28.9,%) towards the (S)-(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad had migrated to a position further along the loop that connects ,7 and ,E; this shows that the position of the catalytic acid is not necessarily conserved in this lipase. [source] | |||||||||||||||||||||||||||||||