LB Agar Plates Research Articles

Silver nanoparticle-coated suture effectively reduces inflammation and improves mechanical strength at intestinal anastomosis in mice

BackgroundOur previous studies have revealed that silver nanoparticles (AgNPs) had anti-inflammatory properties. In this study, we coated AgNPs onto the surface of absorbable suture, to further explore their anti-inflammatory efficacy and potential clinical application using an intestinal anastomosis model. MethodsLayer-by-layer deposition was used to coat AgNPs on absorbable sutures. Scanning electron microscopy (SEM) was conducted to observe the morphology and distribution of AgNPs on suture surface. 1cm of either non-coated suture, suture coated with antibiotics or AgNPs-coated suture was placed on E. coli overlay of LB agar plates to test for bacterial inhibition. The respective sutures were then used for ileal anastomosis in mice. The anastomotic sites were harvested to investigate the degree of tissue inflammation and cell proliferation, as well as collagen deposition. Furthermore, burst pressure measurement was employed to test for mechanical properties. ResultsSEM observation indicated AgNPs could be immobilized and distributed on suture surface evenly. AgNPs-coated suture had the best in vitro anti-bacterial efficacy when compared with other groups. Subsequent immunohistochemistry in the intestinal anastomosis model showed significantly less inflammatory cell infiltration (macrophage and neutrophil) and better collagen deposition in the anastomotic tissue in the AgNPs-coated suture group. Burst pressure measurement in healed anastomosis further confirmed that AgNPs-coated suture had better mechanical properties. ConclusionOur study suggests that AgNPs-coated sutures can improve anastomosis healing due to better mechanical properties from reduced inflammation.

Resistance to AFN-1252 Arises from Missense Mutations in Staphylococcus aureus Enoyl-acyl Carrier Protein Reductase (FabI)

AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 Ki(app) increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate Ki measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step.

Bacillus panacisoli sp. nov., isolated from ginseng soil

A Gram-staining-positive, motile, facultatively anaerobic, endospore-forming and rod-shaped bacterium, designated strain CJ32(T), was isolated from ginseng soil at Geumsan in Korea. The isolate grew optimally at 30 °C, 2% (w/v) NaCl and pH 7.0. Colonies of strain CJ32(T) were beige and circular with an entire margin on LB agar plates. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CJ32(T) was associated with the genus Bacillus and was most closely related to Bacillus graminis YC6957(T) (97.3% similarity) and Bacillus lentus IAM 12466(T) (97.1%). DNA-DNA hybridization with closely related strains was below 31.3%. The major respiratory isoprenoid quinone was MK-7. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The polar lipid profile of strain CJ32(T) consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several unidentified lipids, including phospholipids, aminolipids and aminophospholipids. The predominant fatty acids of strain CJ32(T) were iso-C15:0 and anteiso-C15:0. The G+C content of the genomic DNA was 35.1 mol%. Based on phenotypic, genotypic and phylogenetic data, strain CJ32(T) should be classified within a novel species of the genus Bacillus, for which the name Bacillus panacisoli sp. nov. is proposed. The type strain is strain CJ32(T) ( = KACC 17503(T) = JCM 19226(T)).

Antibiotic resistance of Shewanella putrefaciens isolated from shellfish collected from the West Sea in Korea

In this study, we isolated and characterized Shewanella putrefaciens from shellfish harvested from the West Sea in Korea. For the initial isolation of S. putrefaciens, LB agar plates supplemented with ferrous sulfate and sodium thiosulfate were inoculated with shellfish homogenates, incubated for 24h, and then black colonies were selected. Gram-negative and catalase-positive colonies were subsequently confirmed by PCR assays and API 20E kit test system. The Shewanella-specific 16S rRNA and gyrB genes were used to design S. putrefaciens-specific PCR primers. From 6 species of shellfish tested, 24 S. putrefaciens strains were isolated. These 24 isolates had the following profiles of resistance against 16 antibiotics: all the isolates were resistant to cephalothin and vancomycin and 95.8% were resistant to ampicillin. Here, we report the isolation of S. putrefaciens from shellfish and our results point to a new possible route for exposing healthy individuals to S. putrefaciens.

Myroides guanonis sp. nov., isolated from prehistoric paintings

A novel psychrotolerant, strictly aerobic, non-motile, rod-shaped bacterial strain, designated IM13(T), was isolated from a sample taken from prehistoric guano paintings in Magura Cave, northwest Bulgaria and subjected to a polyphasic taxonomic study. Strain IM13(T) formed yellow colonies on LB agar plates and was Gram-staining-negative, heterotrophic and alkalitolerant. It grew optimally at pH 7.5 and 30 °C in the absence of NaCl. Phylogenetic analysis of the whole 16S rRNA gene revealed that strain IM13(T) branched with representatives of the genus Myroides with sequence similarity of 93-94 % with other species of the genus. The novel isolate contained iso-C15 : 0 (49.1 %), iso-C17 : 1ω9c (18.2 %) and iso-C17 : 0 3-OH (14.0 %) as dominant fatty acids. The DNA G+C content of strain IM13(T) was 33.5 mol%. Based on phylogenetic inference and phenotypic characteristics, it was concluded that strain IM13(T) represents a novel species of the genus Myroides, for which the name Myroides guanonis sp. nov. is proposed. The type strain is IM13(T) ( = DSM 26542(T) = NBIMCC 8736(T)).

Use of antibody gene library for the isolation of specific single chain antibodies by ampicillinantigen conjugates (P3378)

Abstract Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cellsurface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (antifluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single-chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.

Mycobacterium tuberculosis MutT1 (Rv2985) and ADPRase (Rv1700) Proteins Constitute a Two-stage Mechanism of 8-Oxo-dGTP and 8-Oxo-GTP Detoxification and Adenosine to Cytidine Mutation Avoidance

Approximately one third of the world population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. A better understanding of the pathogen biology is crucial to develop new tools/strategies to tackle its spread and treatment. In the host macrophages, the pathogen is exposed to reactive oxygen species, known to damage dGTP and GTP to 8-oxo-dGTP and 8-oxo-GTP, respectively. Incorporation of the damaged nucleotides in nucleic acids is detrimental to organisms. MutT proteins, belonging to a class of Nudix hydrolases, hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the corresponding nucleoside monophosphates and sanitize the nucleotide pool. Mycobacteria possess several MutT proteins. However, a functional homolog of Escherichia coli MutT has not been identified. Here, we characterized MtuMutT1 and Rv1700 proteins of M. tuberculosis. Unlike other MutT proteins, MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 then converts them to the corresponding nucleoside monophosphates. This observation suggests the presence of a two-stage mechanism of 8-oxo-dGTP/8-oxo-GTP detoxification in mycobacteria. MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP with a Km of ∼50 μM and Vmax of ∼0.9 pmol/min per ng of protein, and Rv1700 converts 8-oxo-dGDP to 8-oxo-dGMP with a Km of ∼9.5 μM and Vmax of ∼0.04 pmol/min per ng of protein. Together, MtuMutT1 and Rv1700 offer maximal rescue to E. coli for its MutT deficiency by decreasing A to C mutations (a hallmark of MutT deficiency). We suggest that the concerted action of MtuMutT1 and Rv1700 plays a crucial role in survival of bacteria against oxidative stress.

Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.

Paenibacillus sp. CK214의 swarming 운동성에 미치는 glucose의 영향

Paenibacillus is a gram-positive, spore-forming aerobes that was previously classified as a Bacillus species. Paenibacillus sp. CK214 was highly motile on LB agar plates and showed typical colonial morphology of Paenibacillus. However, its motility was defective in the absence of glucose. Electron microscopic observation revealed that the cells of CK214 cultured on LB agar plates were peritrichously flagellated but not flagellated in the presence of glucose. Flagellar filaments were purified by centrifugation after shearing off from the CK214 cells with vigorous pipetting. The purified protein was composed of a single flagellin with an apparent molecular size of 29 kDa. Recognition of the protein by anti-Edwardsiella tarda flagellin protein antibody demonstrates that the protein is a flagellin protein. A decreased level of flagellin protein was detected in CK214 cells grown under glucose-supplemented media.

Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin–antigen conjugates

Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin–ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.

Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli

Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni-NTA (Ni(2+)-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200ng/ml.

Monitoring the heat stress response of Escherichia coli via NiO nanoparticle assisted MALDI–TOF mass spectrometry

The heat stress response of Escherichia coli at various temperatures has been investigated using NiO nanoparticles assisted MALDI–TOF-MS. Significant numbers of protein peaks were obtained in the presence of NiO NPs when the samples were incubated at various temperatures in comparison with the control E. coli suspension (107cfu/mL). The 10kDa chaperonin (groES) is the principal protein operating both for the protection of proteins from denaturation and in the assembly of newly synthesized proteins. During the heat stress response with NiO NPs, 10kDa chaperonin (grosES) proteins were detected using MALDI–TOF MS. The viability of E. coli was checked on LB agar plates at different temperatures and time treatments. In the presence of NiO NPs, viability decreases drastically; this has been explored and correlated with the MALDI–TOF MS results. Further, surface morphological changes of E. coli at different temperatures were investigated with NiO NPs by transmission electron microscopy (TEM). The response of heat stress toward E. coli for generating more stable protein ions can be applied for bacterial detection under high temperature conditions from biological, clinical and environmental samples.

Reexamining Transcriptional Regulation of the Bacillus subtilis htpX Gene and the ykrK Gene, Encoding a Novel Type of Transcriptional Regulator, and Redefining the YkrK Operator

HtpX is an integral cytoplasmic membrane metalloprotease well conserved in numerous bacteria. A recent study showed that expression of the Bacillus subtilis htpX gene is under dual negative control by Rok and a novel type of transcriptional regulator, YkrK. Here we report that expression of the B. subtilis htpX gene is strongly heat inducible. Contrary to the previous prediction, ykrK expression has been found to be not subject to autoregulation. We have identified the htpX promoter and the authentic ykrK promoter, which is also distinct from the previously predicted one. We have redefined a conserved inverted repeat sequence to be the YkrK operator, which is somewhat different from the previously proposed one. We provide evidence that YkrK is not a substrate of HtpX and that heat induction of htpX is not YkrK mediated. We have also found that the absence of FtsH or HtpX alone did not impair B. subtilis cell viability on LB agar plates at high temperature, whereas the absence of both FtsH and HtpX caused a severe growth defect under heat stress. This finding supports the notion that FtsH and HtpX may have partially overlapping functions in heat resistance. Finally, we show that htpX expression is subject to transient negative control by sigB under heat stress in a Rok- and YkrK-independent manner. Triple negative control of htpX expression at high temperature by rok, sigB, and ykrK may help cells to prevent uncontrolled and detrimental oversynthesis of the HtpX protease.

CONSTRUCTION AND IDENTIFY OF PROKARYOTIC VECTOR OF TIMP-3

CONSTRUCTION AND IDENTIFY OF PROKARYOTIC VECTOR OF TIMP-3

Bacillus subtilis NC1 유래 cellulase와 xylanase의 특성 규명 및 효소 유전자의 규명

Carboxymethylcellulose (CM-cellulose)와 Beechwood xylan을 각각 기질로 사용하여 trypan blue를 첨가하여 제작한 Agar-LB 배지 상에서 명확한 활성환을 형성하는 균주를 cellulase와 xylanase 생산 균주로 단리하였다. 단리한 균주 유래의 16S rRNA 유전자 및 API 50 kit를 분석한 결과 Bacillus subtilis와 약 99.5%의 높은 상동성을 보였기에 본 균주를 Bacillus subtilis로 동정하여 B. subtilis NC1로 명명하였다. B. subtilis NC1 유래 cellulase와 xylanase는 CM-cellulose와 Beechwood xylan에 대하여 각각 높은 효소 활성을 보였으며, 두 효소 모두 pH 5.0과 <TEX>$50^{\circ}C$</TEX>의 조건하에서 가장 높은 효소 활성을 보였다. B. subtilis NC1 균주 유래 cellulase와 xylanase 유전자를 cloning하기 위하여 shot-gun cloning 방법을 이용하여 B. subtilis NC1 염색체 DNA로부터 효소 유전자를 cloning하여 유전자 배열을 규명한 결과 cellulase 유전자는 아미노산 499개를 암호화하는 1,500 bp의 open reading frame (ORF)으로 이루어져 있었으며, 아미노산 배열로부터 추정되는 분자량은 55,251 Da 이었다. 그리고, xylanase에 대한 유전자는 아미노산 422개를 암호화하는 1,269 bp의 ORF로 이루어져 있었으며 유전자 유래 아미노산 배열로부터 추정되는 단백질 분자량은 47,423 Da 이었다. 두 효소의 아미노산 배열을 이용하여 상동성을 검토한 결과 cellulase는 glycoside hydrolase family (GH) 5에 속하는 cellulase와 xylanase는 GH30에 속하는 xylanase와 높은 상동성을 나타내었다. A Bacillus sp. strain producing celluase and xylanase was isolated from environmental soil with LB agar plate containing carboxymethylcellulose (CM-cellulose) and beechwood xylan stained with trypan blue as substrates, respectively. Based on the 16S rRNA gene sequence and API 50 CHL test, the strain was identified as B. subtilis and named B. subtilis NC1. The cellulase and xylanase from B. subtilis NC1 exhibited the highest activities for CM-cellulose and beechwood xylan as substrate, respectively, and both enzymes showed the maximum activity at pH 5.0 and <TEX>$50^{\circ}C$</TEX>. We cloned and sequenced the genes for cellulase and xylanase from genomic DNA of the B. subtilis NC1 by the shot-gun cloning method. The cloned cellulase and xylanase genes consisted of a 1,500 bp open reading frame (ORF) encoding a 499 amino acid protein with a calculated molecular mass of 55,251 Da and a 1,269 bp ORF encoding a 422 amino acid protein with a calculated molecular mass of 47,423 Da, respectively. The deduced amino acid sequences from the genes of cellulase and xylanase showed high identity with glycosyl hydrolases family (GH) 5 and 30, respectively.

Characterization of a lipopeptide biosurfactant produced by a crude-oil-emulsifying Bacillus sp. I-15

Petroleum-polluted environmental samples were collected to isolate and characterize biosurfactant-producing bacteria. Culturing of the collected samples in LB-broth and enrichments in chemically defined medium (CDM) with various carbon sources yielded 51 strains, some of which produced biosurfactants/bioemulsifiers when challenged with crude oil as a sole carbon source. The I-15 isolate, a Gram-positive, motile bacillus, emulsified crude oil in CDM without reduction in surface tension. Compared to LB-grown cells, the crude oil-grown I-15 cells were surrounded by a transparent zone and produced heavily mucoid colonies on LB-agar plates. When grown with glucose as a sole carbon source, the I-15 strain produced a biosurfactant extracellularly, resulting in a 65% reduction in surface tension of the growth medium. The glucose-grown cells were coccoid to irregular in shape. Biosurfactant production was concomitant with reversible growth decline interrupting the exponential phase. Infrared spectroscopy and thin layer chromatography suggested a lipopeptide structure for the crude biosurfactant. Partial sequences of 10 16S rDNA gene clones from the I-15 strain were highly similar to those of various members of the family Bacillaceae. The I-15 strain is a promising biosurfactant producer and is probably an active indigenous crude oil degrader. Biosurfactant production is accompanied by morphological and physiological alterations. The I-15 strain possesses intragenomic heterogeneity in the rrn (RNA) operons.

OC-158 Endogenous production of antibiotics by mesenchymal stem cells and the potential value in crohn's fistula healing

IntroductionPotent effects of human mesenchymal stem cells (MSCs) on immune responses have been demonstrated in vitro studies and animal models, encouraging further investigations into their clinical value. Studies of MSCs...

Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates

Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1-50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04kcal/mol, within a temperature range of 1-40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.