The ability to restore the activity of the apoenzyme and identification by HPLC analysis from the holoenzyme suggests that the enzyme contains FAD as the prosthetic group (Table 4). Loosely bound FAD as the prosthetic group has been reported for several flavin hydroxylases (Takemori et al., 1969; Strickland & Massey, 1973; Elmorsi & Hopper, 1977; Wang et al., 1984; Tanner & Hopper, 2000). The enzyme could accept both NADPH

and NADH as an external electron donor and does not show nonspecific NAD(P)H oxidase activity. External addition of metal ions and chelators has no effect on the activity. The homodimeric nature of the enzyme (subunit molecular weight of 34 kDa) suggests that 1-hydroxy-2-naphthoic acid hydroxylase is a FAD-containing single-component Veliparib solubility dmso hydroxylase. The molecular mass of

the single component system salicylate-1-hydroxylases are reported to be in the range of 38–57 kDa and are either monomers or dimers (Yamamoto et al., 1965; White-Stevens & Kamin, 1972; You et al., 1990; Balashova et al., 2001). A three-component salicylate-1-hydroxylase consisting of an oxygenase, a ferredoxin and a reductase has also been reported (Pinyakong Histone Methyltransferase inhibitor et al., 2003; Jouanneau et al., 2007). Flavin hydroxylases have been reported to accept electrons from NADH, NADPH or both (Ohta & Ribbons, 1976; Beadle & Smith, 1982; Van Berkel & Van Den Tweel, 1991; Swetha et al., 2007). Similarly, 1-hydroxy-2-naphthoic acid hydroxylase accepted electrons from both NADPH and NADH. The kinetic constants for NADPH or NADH clearly indicate that both electron donors are equally preferred by the enzyme (Table 5). The affinity for 1-H2NA (Km) remained unchanged, irrespective of the electron donor

used. The enzyme saturation profiles with 1-H2NA, NADPH or NADH were sigmoidal, suggesting a regulatory role of this enzyme in the phenanthrene degradation pathway. A similar kinetic property has been reported for 3-hydroxybenzoate 6-hydroxylase from Cediranib (AZD2171) Klebsiella pneumoniae (Suarez et al., 1995), but not for salicylate hydroxylases so far. 1-Hydroxy-2-naphthoic acid hydroxylase from strain PPH failed to show the conversion of 1-H2NA to 1,2-DHN under anaerobic conditions, suggesting that the enzyme belongs to the oxygenase group. A majority of flavin hydroxylases, including salicylate hydroxylases, have been reported to be exhibiting broad substrate specificity (Beadle & Smith, 1982; Locher et al., 1991; Xun et al., 1992; Suske et al., 1997; Eppink et al., 2000). 1-Hydroxy-2-naphthoic acid hydroxylase from strain PPH was specific to 1-H2NA and failed to show activity on 1-H2NA analogs and salicylate. Flavoprotein hydroxylases with limited substrate have also been reported (Hosokawa & Stanier, 1966; Van Berkel & Van Den Tweel, 1991; Suarez et al., 1995; Haigler et al., 1996; Swetha et al., 2007).

hydrophila J-1 and NJ-4 cultures mixed with an equal volume of PYG medium and T. thermophila BF1 cell suspensions without bacteria served as controls. An equal volume of LB and PYG mixture

was used as the blank. The plate was incubated overnight at 30 °C. The growth of bacteria was determined by measuring the changes of OD450 nm. Tetrahymena cells only accounted for a negligible absorbance (Benghezal www.selleckchem.com/screening/chemical-library.html et al., 2007). The starter culture of T. thermophila BF1 was cultured at 30 °C overnight and 5 mL was used to inoculate 100 mL fresh PYG in a 250-mL Erlenmeyer flask for 48 h at 30 °C without shaking. Tetrahymena thermophila were then starved following centrifugation at 2000 g for 10 min at 15 °C, washed in PBSS (2 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2 and pH 7.0 adjusted with Tris) once and maintained in PBSS for 12 h at 30 °C without shaking. Tetrahymena thermophila BF1 were counted using a hemacytometer and then diluted in PBSS to a concentration of 105 cells mL−1. Twenty-five milliliters of T. thermophila BF1 diluted in PBSS was then transferred into a 50-mL centrifuge tube and incubated at 30 °C for 1 h. Aeromonas hydrophila J-1 and NJ-4 at a multiplicity of infection of 100 were added to respective Autophagy inhibitor chemical structure tubes and 1-mL aliquots were collected into 1.5-mL Eppendorf tubes. Two aliquots were examined every 6 h to assess T. thermophila BF1 biomass and the presence of

intracellular bacteria. The T. thermophila BF1 biomass was measured by counting live organisms using a hemacytometer. The number of intracellular A. hydrophila J-1 or NJ-4 was determined using a gentamycin protection assay as follows: 80 μg mL−1 gentamycin in PBSS was added to a 900-μL co-culture for 1 h to kill extracellular bacteria. Samples were then centrifuged at 2000 g for 10 min and the ciliates were collected and washed once in PBSS. The T. thermophila pellet was then resuspended in 900 μL of 1% Triton X-100 for 30 min at 37 °C in order to release intracellular bacteria. The lysates were serially diluted in PBSS and plated on LB agar plates. The number of

bacterial colonies was counted after inoculation FER at 37 °C overnight. A fragment containing the entire green fluorescent protein (GFP) ORF was cloned from pFPV25.1 into the SacI site of the vector pWSK129. This construct, pWSK129-gfp, was subsequently transformed into A. hydrophila J-1, thus producing a GFP-expressing A. hydrophila J-1 (AhJ-1GFP). To assess the intracellular localization of AhJ-1GFP within T. thermophila BF1, starved T. thermophila BF1 cultures were co-cultured with AhJ-1GFP for 4–5 h at 30 °C and examined using a fluorescence microscope (Zeiss). Tetrahymena thermophila BF1 not incubated with AhJ-1GFP was used as the negative control. After T. thermophila BF1 were co-cultured with A. hydrophila J-1 in LB for 2 h, the ciliates were pelleted and immediately fixed with 2.5% glutaraldehyde.

The suppression of action potentials was preserved under blockade of postsynaptic G-proteins, although baclofen-induced hyperpolarisation

selleckchem was completely blocked. These findings suggest presynaptic effects of baclofen on the induced action potentials. Under voltage-clamp conditions, application of baclofen reduced the frequency, but not the amplitude, of miniature excitatory postsynaptic currents (mEPSCs), whereas the GABAB receptor antagonist CGP55845 increased the frequency of mEPSCs without affecting the amplitude. Furthermore, application of a GABA uptake inhibitor, nipecotic acid, decreased the frequency of mEPSCs; this effect was blocked by CGP55845, but not by the GABAA antagonist bicuculline. Both the frequency and the amplitude of the pinch-evoked barrage of excitatory postsynaptic currents (EPSCs) were suppressed by baclofen

in a dose-dependent GSK2118436 manner. The frequency and amplitude of touch-evoked EPSCs was also suppressed by baclofen, but the suppression was significantly smaller than that of pinch-evoked EPSCs. We conclude that mechanical noxious transmission is presynaptically blocked through GABAB receptors in the SG, and is more effectively suppressed than innocuous transmission, which may account for a part of the mechanism of the efficient analgesic effects of baclofen. “
“The N-methyl-d-aspartate receptor (NMDAR) exhibits strong voltage-dependent block by extracellular Mg2+, which is relieved by sustained depolarization and glutamate binding, and which is central to the function of the NMDAR

in synaptic plasticity. Rapid membrane depolarization during agonist application reveals a slow unblock of NMDARs, which has important functional implications, for example in the generation of NMDAR spikes, and in determining the narrow time window for spike-timing-dependent plasticity. However, its mechanism is still unclear. Here, we study unblock of divalent cations in native NMDARs in nucleated patches isolated from mouse cortical layer 2/3 pyramidal neurons. Comparing unblock kinetics of NMDARs in the presence of extracellular Mg2+or in nominally zero Mg2+, and with Mn2+or Co2+substituting for Mg2+, we found that the properties of slow unblock Idelalisib chemical structure were determined by the identity of the blocking metal ion at the binding site, presumably by affecting the operation of a structural link to channel gating. The time course of slow unblock was not affected by zinc, or the zinc chelator TPEN [N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine], while the slower fraction of unblock was reduced by ifenprodil, an NR2B-selective antagonist. Slow unblock was only weakly temperature dependent, speeding up with rise in temperature with a Q10 of ≈1.5. Finally, using action potential waveform voltage-clamp, we show that this slow relief from divalent cation block is a prominent feature in physiologically realistic patterns of changing membrane potential.

putida PCL1445 (data not shown). The stability of the plasmids and the transposon integration was

tested by subculturing in nonselective media (without antibiotic selection pressure) for approximately 30 generations. Samples of the subcultures were plated and colonies were screened for the expression of mcherry by fluorescence microscopy. Strain PCL1481 carrying miniTn7∷mcherry did not show any loss of integration. No loss of plasmid was observed for PCL1479 carrying pMP7604, whereas 3% of the colonies of strain PCL1480 carrying pMP7605 had lost fluorescence at day 3 (data not shown). A qualitative and quantitative analysis for mCherry production in P. putida PCL1445 tagged with pMP7604, pMP7605 and pMP7607 was performed in order to evaluate the resulting brightness of the different Volasertib research buy constructs. Cells of overnight cultures were visualized using fluorescence and light microscopy (Fig. 3a) and fluorescence was quantified using fluorometry (Fig. 2b). mcherry expression was detected at the single-cell level for all tagged strains. Microscopic and fluorometric analyses showed that strain PCL1480 (harboring pMP7605) produced the highest amount of mCherry and strain PCL1481 (containing miniTn7-mcherry)

produced the lowest amount (Fig. 3a and b). The strains PCL1479, PCI-32765 manufacturer PCL1480 and PCL1481 produced mCherry in a ratio of 15 : 95 : 1, respectively. No significant fluorescence was detected for P. putida PCL1445 cells and strains PCL1477 and PCL1478 containing the cloning vectors pME6031 and pBBR1MCS-5 (Fig. 2b). To evaluate the applicability of the mCherry marker vectors for tagging Gram-negative bacteria, several other Gram-negative spp., such as P. fluorescens WCS365 (an efficient root colonizer), P. aeruginosa PAO1 (a model strain for cystic fibrosis research)

and E. tarda FL6-60 (a fish pathogen and model for zebrafish immunology), were transformed with pMP7604 and pMP7605. This yielded PCL1700, PCL1701, PCA0241, PCA0242, PCA0239 and PCA0240, respectively. Fluorescence microscopy analysis showed the production of mCherry for all transformed strains (data not shown). Single colonies were isolated and overnight cultures were grown for quantitative analysis of mCherry production and comparison with P. putida PCL1445 (Fig. 4). Strains containing pMP7605 showed the highest mCherry medroxyprogesterone production. Comparable mCherry production levels were observed among the four strains tested, except for the one carrying pMP7605, which showed a lower level of expression in E. tarda FL6-60. To analyze the applicability of the mcherry-expressing constructs pMP7604, pMP7605 and pMP7607 in established test systems, which are not suitable for efficient application of antibiotic pressure, P. putida PCL1445-tagged strains were allowed to form biofilms on glass (in vitro biofilm assay) and on tomato roots (in vivo assay used to study root colonization). Using CLSM, the tagged strains were visualized at the single-cell level in both assays (Fig.

putida PCL1445 (data not shown). The stability of the plasmids and the transposon integration was

tested by subculturing in nonselective media (without antibiotic selection pressure) for approximately 30 generations. Samples of the subcultures were plated and colonies were screened for the expression of mcherry by fluorescence microscopy. Strain PCL1481 carrying miniTn7∷mcherry did not show any loss of integration. No loss of plasmid was observed for PCL1479 carrying pMP7604, whereas 3% of the colonies of strain PCL1480 carrying pMP7605 had lost fluorescence at day 3 (data not shown). A qualitative and quantitative analysis for mCherry production in P. putida PCL1445 tagged with pMP7604, pMP7605 and pMP7607 was performed in order to evaluate the resulting brightness of the different selleck inhibitor constructs. Cells of overnight cultures were visualized using fluorescence and light microscopy (Fig. 3a) and fluorescence was quantified using fluorometry (Fig. 2b). mcherry expression was detected at the single-cell level for all tagged strains. Microscopic and fluorometric analyses showed that strain PCL1480 (harboring pMP7605) produced the highest amount of mCherry and strain PCL1481 (containing miniTn7-mcherry)

produced the lowest amount (Fig. 3a and b). The strains PCL1479, http://www.selleckchem.com/products/azd2014.html PCL1480 and PCL1481 produced mCherry in a ratio of 15 : 95 : 1, respectively. No significant fluorescence was detected for P. putida PCL1445 cells and strains PCL1477 and PCL1478 containing the cloning vectors pME6031 and pBBR1MCS-5 (Fig. 2b). To evaluate the applicability of the mCherry marker vectors for tagging Gram-negative bacteria, several other Gram-negative spp., such as P. fluorescens WCS365 (an efficient root colonizer), P. aeruginosa PAO1 (a model strain for cystic fibrosis research)

and E. tarda FL6-60 (a fish pathogen and model for zebrafish immunology), were transformed with pMP7604 and pMP7605. This yielded PCL1700, PCL1701, PCA0241, PCA0242, PCA0239 and PCA0240, respectively. Fluorescence microscopy analysis showed the production of mCherry for all transformed strains (data not shown). Single colonies were isolated and overnight cultures were grown for quantitative analysis of mCherry production and comparison with P. putida PCL1445 (Fig. 4). Strains containing pMP7605 showed the highest mCherry Florfenicol production. Comparable mCherry production levels were observed among the four strains tested, except for the one carrying pMP7605, which showed a lower level of expression in E. tarda FL6-60. To analyze the applicability of the mcherry-expressing constructs pMP7604, pMP7605 and pMP7607 in established test systems, which are not suitable for efficient application of antibiotic pressure, P. putida PCL1445-tagged strains were allowed to form biofilms on glass (in vitro biofilm assay) and on tomato roots (in vivo assay used to study root colonization). Using CLSM, the tagged strains were visualized at the single-cell level in both assays (Fig.

After the growth proceeded to opacity, an aliquot was removed and used as an inoculum in a new bottle with fresh medium and an additional 5% ethanol for another 12-h incubation. The cultures were diluted and spread on agar plates without ethanol. Individual colonies, visible after 8–10 h, were subcultured in medium supplemented with 10% ethanol to confirm the ethanol tolerance of the strains. The strains were purified by repeatedly isolating single colonies in agar plates at least five times. The type strains of A. flavithermus DSM 2641T, Anoxybacillus pushchinoensis DSM 12423T and Anoxybacillus kestanbolensis NCIMB 13971T were obtained from the Deutsche Sammlung von Mikroorganismen

H 89 mw und Zellkulturen (DSMZ). Anoxybacillus eryuanensis KCTC 13720T and Anoxybacillus tengchongensis KCTC 13721T were acquired from the Korean Collection for Type Cultures (KCTC). The cells were initially cultured overnight in LB medium with 4% ethanol. The culture was washed

twice in unsupplemented medium to remove ethanol and then used for inoculation of medium with ethanol added at concentrations Talazoparib solubility dmso of 0–10%. The cultures were incubated without shaking at temperatures in the range of 45–65 °C for 60 h. Samples for measurement were withdrawn directly from the sealed bottles with sterile syringes before and after incubation. Growth was measured spectrophotometrically at 600 nm. To evaluate whether this organism can metabolize ethanol, ethanol was analyzed by Agilent 7890A GC System equipped with an Agilent 7694E Headspace Sampler (Agilent Technologies). The microbial biofilm and its formation were observed by light microscopy (Olympus, BX51). Sterile glass slides were placed in LB culture supplemented with 13% ethanol. The culture was incubated without shaking at 60 °C for 24 h. The slides were then taken out of the bottles and washed

three times with H2O. The remaining cells were fixed with methanol for 10 min and stained with 2% (w/v) crystal violet for 5 min. Physiological and biochemical tests were carried out without ethanol addition at 60 °C. Conventional biochemical tests were performed according to standard methods (Smibert & Krieg, 1994). Anaerobic growth experiments were carried out in Hungate tubes. The ability to utilize Thalidomide different carbon source was examined in basal medium (Pikuta et al., 2000). To minimize the effects of growth temperature and different media on bacterial fatty acid composition, all strains were uniformly incubated at 60 °C for 24 h on agar LB medium. Then, the analysis of cellular fatty acid methyl esters were performed according to the method described in the Sherlock Microbial Identification System manual (version4.0, MIDI). The final extracts were analyzed by GC/MS in scan mode, using an Agilent 7890 GC/5975 MSD system (Agilent Technologies).

After the growth proceeded to opacity, an aliquot was removed and used as an inoculum in a new bottle with fresh medium and an additional 5% ethanol for another 12-h incubation. The cultures were diluted and spread on agar plates without ethanol. Individual colonies, visible after 8–10 h, were subcultured in medium supplemented with 10% ethanol to confirm the ethanol tolerance of the strains. The strains were purified by repeatedly isolating single colonies in agar plates at least five times. The type strains of A. flavithermus DSM 2641T, Anoxybacillus pushchinoensis DSM 12423T and Anoxybacillus kestanbolensis NCIMB 13971T were obtained from the Deutsche Sammlung von Mikroorganismen

Selleckchem EPZ015666 und Zellkulturen (DSMZ). Anoxybacillus eryuanensis KCTC 13720T and Anoxybacillus tengchongensis KCTC 13721T were acquired from the Korean Collection for Type Cultures (KCTC). The cells were initially cultured overnight in LB medium with 4% ethanol. The culture was washed

twice in unsupplemented medium to remove ethanol and then used for inoculation of medium with ethanol added at concentrations www.selleckchem.com/products/wnt-c59-c59.html of 0–10%. The cultures were incubated without shaking at temperatures in the range of 45–65 °C for 60 h. Samples for measurement were withdrawn directly from the sealed bottles with sterile syringes before and after incubation. Growth was measured spectrophotometrically at 600 nm. To evaluate whether this organism can metabolize ethanol, ethanol was analyzed by Agilent 7890A GC System equipped with an Agilent 7694E Headspace Sampler (Agilent Technologies). The microbial biofilm and its formation were observed by light microscopy (Olympus, BX51). Sterile glass slides were placed in LB culture supplemented with 13% ethanol. The culture was incubated without shaking at 60 °C for 24 h. The slides were then taken out of the bottles and washed

three times with H2O. The remaining cells were fixed with methanol for 10 min and stained with 2% (w/v) crystal violet for 5 min. Physiological and biochemical tests were carried out without ethanol addition at 60 °C. Conventional biochemical tests were performed according to standard methods (Smibert & Krieg, 1994). Anaerobic growth experiments were carried out in Hungate tubes. The ability to utilize RAS p21 protein activator 1 different carbon source was examined in basal medium (Pikuta et al., 2000). To minimize the effects of growth temperature and different media on bacterial fatty acid composition, all strains were uniformly incubated at 60 °C for 24 h on agar LB medium. Then, the analysis of cellular fatty acid methyl esters were performed according to the method described in the Sherlock Microbial Identification System manual (version4.0, MIDI). The final extracts were analyzed by GC/MS in scan mode, using an Agilent 7890 GC/5975 MSD system (Agilent Technologies).

After the growth proceeded to opacity, an aliquot was removed and used as an inoculum in a new bottle with fresh medium and an additional 5% ethanol for another 12-h incubation. The cultures were diluted and spread on agar plates without ethanol. Individual colonies, visible after 8–10 h, were subcultured in medium supplemented with 10% ethanol to confirm the ethanol tolerance of the strains. The strains were purified by repeatedly isolating single colonies in agar plates at least five times. The type strains of A. flavithermus DSM 2641T, Anoxybacillus pushchinoensis DSM 12423T and Anoxybacillus kestanbolensis NCIMB 13971T were obtained from the Deutsche Sammlung von Mikroorganismen

Ku-0059436 molecular weight und Zellkulturen (DSMZ). Anoxybacillus eryuanensis KCTC 13720T and Anoxybacillus tengchongensis KCTC 13721T were acquired from the Korean Collection for Type Cultures (KCTC). The cells were initially cultured overnight in LB medium with 4% ethanol. The culture was washed

twice in unsupplemented medium to remove ethanol and then used for inoculation of medium with ethanol added at concentrations selleck screening library of 0–10%. The cultures were incubated without shaking at temperatures in the range of 45–65 °C for 60 h. Samples for measurement were withdrawn directly from the sealed bottles with sterile syringes before and after incubation. Growth was measured spectrophotometrically at 600 nm. To evaluate whether this organism can metabolize ethanol, ethanol was analyzed by Agilent 7890A GC System equipped with an Agilent 7694E Headspace Sampler (Agilent Technologies). The microbial biofilm and its formation were observed by light microscopy (Olympus, BX51). Sterile glass slides were placed in LB culture supplemented with 13% ethanol. The culture was incubated without shaking at 60 °C for 24 h. The slides were then taken out of the bottles and washed

three times with H2O. The remaining cells were fixed with methanol for 10 min and stained with 2% (w/v) crystal violet for 5 min. Physiological and biochemical tests were carried out without ethanol addition at 60 °C. Conventional biochemical tests were performed according to standard methods (Smibert & Krieg, 1994). Anaerobic growth experiments were carried out in Hungate tubes. The ability to utilize Methocarbamol different carbon source was examined in basal medium (Pikuta et al., 2000). To minimize the effects of growth temperature and different media on bacterial fatty acid composition, all strains were uniformly incubated at 60 °C for 24 h on agar LB medium. Then, the analysis of cellular fatty acid methyl esters were performed according to the method described in the Sherlock Microbial Identification System manual (version4.0, MIDI). The final extracts were analyzed by GC/MS in scan mode, using an Agilent 7890 GC/5975 MSD system (Agilent Technologies).

In fact, an inner KT protein Ndc10 plays the central role in S. cerevisiae (Fig. 2a), while the middle KT proteins – Mis6 and Spc7 – play governing roles to a great extent in S. pombe (Fig. 2b). This process is remarkably diverged with a complex interdependence among many essential KT proteins from various layers in C. albicans (Fig. 2c). Unravelling this fascinating molecular

mechanism of KT assembly in many organisms will improve our understanding of how the KT assembly pathways coevolved with the CEN DNA during speciation. We thank B. Suma (Central instrumentation facility, Molecular Lapatinib purchase Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research) for confocal microscopy and image processing. We are thankful to the members of Sanyal laboratory for insightful comments. We express our regret to our colleagues whose work could not be cited due to space limitations. “
“Human milk oligosaccharides (HMO) are prominent among the functional components of human breast milk. While HMO have potential applications in both infants and adults, this potential is limited by the difficulties Rapamycin cell line in manufacturing these complex structures. Consequently, functional alternatives such as galacto-oligosaccharides are under investigation, and nowadays, infant formulae are supplemented with galacto-oligosaccharides

to mimic the biological effects of HMO. Recently, approaches toward the production of defined human milk oligosaccharide structures using microbial, fermentative methods employing single, appropriately engineered microorganisms Acetophenone were introduced. Furthermore, galactose-containing hetero-oligosaccharides have attracted an increasing amount of attention because they are structurally more closely related to HMO. The synthesis of these novel oligosaccharides, which resemble the core of HMO, is of great interest for

applications in the food industry. “
“The polymerization of free nucleotides into new genetic elements by DNA polymerases in the absence of DNA, called ab initio DNA synthesis, is a little known phenomenon. DNA polymerases from prokaryotes can effectively synthesize long stretches of linear double-stranded DNA in the complete absence of added primer and template DNAs. Ab initio DNA synthesis is extremely enhanced if a restriction endonuclease or nicking endonuclease is added to the reaction with DNA polymerase. The synthesized ab initio DNA have various tandem repeats. Sequences similar to those of ab initio DNA products are found in many natural genes. The significance of ab initio DNA synthesis is that genetic information can be created directly by protein. The ab initio DNA synthesis is considered a non-specific synthesis in various DNA amplification techniques. In this review, we present the main studies devoted to this phenomenon and introduce possible mechanisms of this synthesis from our current knowledge.

, 2008), is the direct regulation of molecular target(s) modulating the flocculation behavior, then mutations that impair CheA1 or CheY1 functions should yield similar phenotypes. This study revealed several distinguishing features of the flocs formed by each of the mutant strains that are not consistent with a direct function of Che1 BMS354825 in the regulation of flocculation. First, although cells of both mutant strains were adherent and embedded in a complex matrix apparently comprised of fibrillar material, cell-to-cell contacts within the matrix of the AB102 (ΔcheY1) strain were separated by a

thick layer that was visible by AFM after 1 week. This layer formed a tight network around each individual cell within the floc. In contrast, in the flocs of AB101 (ΔcheA1), individual cells were distinctly defined and no obvious connecting features were observed between the cells. Because it is impossible to determine the composition of this material from imaging alone, we used flocculation inhibition and lectin-binding assays to analyze the different

structures observed between the two strains in more detail. The results of the lectin-binding assay click here suggest that AB101 (ΔcheA1) produces an exopolysaccharide that is more abundant in α-mannose and/or α-glucose, and N-acetyl galactosamine than the exopolysaccharide produced by AB102 (ΔcheY1). Previous studies have shown that the glucose content of exopolysaccharide is significantly lower during flocculation in the wild-type Sp7 strain and in other mutant derivative strains with increased aggregation capacity (Bahat-Samet et al., 2004). Consistent with these data, AB102 (ΔcheY1) strain displays a stronger flocculation phenotype and its extracellular matrix appears to have a reduced mannose and/or a glucose content relative to that of AB101 (ΔcheA1). An alternative explanation stiripentol for these data is that the structural organization of the AB102 (ΔcheY1) floc reduces the accessibility of the sugar residues to the lectin, thus limiting the amount of lectin

that binds to the cells and the surrounding matrix. Even though the floc structures of the two mutant strains showed different binding affinities for lectins, indicating possible differences in the polysaccharide composition of the exopolysaccharide produced during flocculation, these results do not necessarily demonstrate the contribution of specific polysaccharides to aggregation or flocculation. Previous studies showed that exopolysaccharide composition is modified over time from a glucose-rich exopolysaccharide to an arabinose-rich exopolysaccharide and that this temporal change correlates directly with the timing of flocculation (Bahat-Samet et al., 2004). In agreement with this observation, flocs formed by the ΔcheY1 mutant were more sensitive to the addition of arabinose in the flocculation inhibition assay, suggesting that the sugar residues comprising the matrix of these strains are different in structure and/or composition.