M9 Minimal Medium Research Articles

METHODS: Optimization of culture medium for production of recombinant dengue protein inEscherichia coli

Enhanced production of recombinant dengue multi-epitope protein in Escherichia coli was achieved by optimization of culture medium. Complex media (Luria Bertani broth, Terrific broth, Super broth, M9 minimal media, five times Luria Bertani broth, semi-defined medium enriched with tryptone and yeast extract, and semi-defined medium enriched with glucose) were evaluated for production of recombinant dengue multi-epitope protein in shake flasks. The recombinant protein was further produced by fed-batch fermentation using 5 L bioreactor. Cells were grown in optimized semi-defined medium, and feeding was carried out with 5X medium and glycerol. When growth reached 14.35 g/L of dry cell weight, culture was induced with 0.5 mM IPTG and further grown for 4 h to reach 18.37 g/L dry cell weight. The recombinant dengue multi-epitope protein was purified from inclusion bodies under denaturing conditions using metal affinity chromatography, which yielded 96.43 mg/L of protein. The purified protein was found to be reactive with dengue-infected human serum samples.

Fermentative hydrogen yields from different sugars by batch cultures of metabolically engineered Escherichia coli DJT135

Future sustainable production of biofuels will depend upon the ability to use complex substrates present in biomass if the use of simple sugars derived from food crops is to be avoided. Therefore, organisms capable of using a variety of fermentable carbon sources must be found or developed for processes that could produce hydrogen via fermentation. Here we have examined the ability of a metabolically engineered strain of Escherichia coli, DJT135, to produce hydrogen from glucose as well as various other carbon sources, including pentoses. The effects of pH, temperature and carbon source were investigated in batch experiments. Maximal hydrogen production from glucose was obtained at an initial pH of 6.5 and temperature of 35 °C. Kinetic growth studies showed that the μmax was 0.0495 h −1 with a Ks of 0.0274 g L −1 when glucose was the sole carbon source in M9 (1X) minimal medium. Among the many sugar and sugar derivatives tested, hydrogen yields were highest with fructose, sorbitol and d-glucose; 1.27, 1.46 and 1.51 mol H 2 mol −1 substrate respectively.

Fibronectin Binds to and Induces Conformational Change in a Disordered Region of Leptospiral Immunoglobulin-like Protein B

Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (K(D) = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (K(av)) measured with gel permeation chromatography, a high hydrodynamic radius (R(h)) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional (15)N-(1)H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a beta-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a beta-strand-rich structure, suggestive of the known beta-zipper mode of NTD binding.

Identification and Quantitation of Newly Synthesized Proteins in Escherichia coli by Enrichment of Azidohomoalanine-labeled Peptides with Diagonal Chromatography

A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhal-labeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively modified peptides are identified by reversed phase liquid chromatography and on-line tandem mass spectrometry. We identified 527 proteins representative of all major Gene Ontology categories. Comparing the relative amounts of 344 proteins synthesized in 15 min upon a switch of growth temperature from 37 to 44 degrees C showed that nearly 20% increased or decreased more than 2-fold. Among the most up-regulated proteins many were chaperones and proteases in accordance with the cells response to unfolded proteins due to heat stress. Comparison of our data with results from previous microarray experiments revealed the importance of regulation of gene expression at the level of transcription of the most elevated proteins under heat shock conditions and enabled identification of several candidate genes whose expression may predominantly be regulated at the level of translation. This work demonstrates for the first time the use of a bioorthogonal amino acid for proteome-wide detection of changes in the amounts of proteins synthesized during a brief period upon variations in cellular growth conditions. Comparison of such data with relative mRNA levels enables assessment of the separate contributions of transcription and translation to the regulation of gene expression.

Cloning of a novel levoglucosan kinase gene from Lipomyces starkeyi and its expression in Escherichia coli

Levoglucosan, cellulosic pyrolysate, is converted to glucose-6-phosphate by a specific levoglucosan kinase in fungi. A novel cDNA of levoglucosan kinase gene (lgk) from yeast Lipomyces starkeyi YZ-215 was isolated by RACE method. The 1,445 bp cDNA fragment (lgk) harbouring the kinase gene exhibited one open reading frame (ORF) composed of 1,317 bp flanked by a 14 bp 5′-UTR and a 114 bp 3′-UTR, including a 25 bp poly(A) tail. The ORF encoded a 439 amino acid polypeptide with a 48.4 kDa predicted molecular mass. Analysis of amino sequence revealed that the kinase belonged to the bacterial anhydro-N-acetylmuramic acid kinase (AnmK) family, and kinase-like proteins existed in some fungi, especially in filamentous fungi such as Aspergillus. The kinase gene was transformed into Escherichia coli BL21 (DE3), recombinant E. coli could grow in M9 minimal medium with levoglucosan as a sole carbon source when induced by IPTG. In addition, the recombinant kinase was overexpressed, purified and characterized. The kinase was stable at pH 7–10 and showed maximum activity at 30°C and pH 9.0 as natural kinase, but presented higher thermostability. Kinetic constants (apparent K m values) for LG and ATP were 105.3 ± 12.5 and 0.20 ± 0.02 mM, respectively. Furthermore, the kinase showed substrate specificity for LG. This novel levoglucosan kinase gene would be useful in constructing recombinant microbial strains for the efficient bioconversion of cellulosic pyrolysate to ethanol.

Microbial responses to xenobiotic compounds. Identification of genes that allow Pseudomonas putida KT2440 to cope with 2,4,6‐trinitrotoluene

SummaryPseudomonas putida KT2440 grows in M9 minimal medium with glucose in the presence of 2,4,6‐trinitrotoluene (TNT) at a similar rate than in the absence of TNT, although global transcriptional analysis using DNA microarrays revealed that TNT exerts some stress. Response to TNT stress is regulated at the transcriptional level, as significant changes in the level of expression of 65 genes were observed. Of these genes, 39 appeared upregulated, and 26 were downregulated. The identity of upregulated genes suggests that P. putida uses two kinds of strategies to overcome TNT toxicity: (i) induction of genes encoding nitroreductases and detoxification‐related enzymes (pnrA, xenD, acpD) and (ii) induction of multidrug efflux pump genes (mexEF/oprN) to reduce intracellular TNT concentrations. Mutants of 13 up‐ and 7 downregulated genes were analysed with regards to TNT toxicity revealing the role of the MexE/MexF/OprN pump and a putative isoquinoline 1‐oxidoreductase in tolerance to TNT. The ORF PP1232 whose transcriptional level did not change in response to TNT affected growth in the presence of nitroaromatic compounds and it was found in a screening of 4000 randomly generated mutants.

Crystal Structure of the Caseinolytic Protease Gene Regulator, a Transcriptional Activator in Actinomycetes

Human pathogens of the genera Corynebacterium and Mycobacterium possess the transcriptional activator ClgR (clp gene regulator) which in Corynebacterium glutamicum has been shown to regulate the expression of the ClpCP protease genes. ClgR specifically binds to pseudo-palindromic operator regions upstream of clpC and clpP1P2. Here, we present the first crystal structure of a ClgR protein from C. glutamicum. The structure was determined from two different crystal forms to resolutions of 1.75 and 2.05 A, respectively. ClgR folds into a five-helix bundle with a helix-turn-helix motif typical for DNA-binding proteins. Upon dimerization the two DNA-recognition helices are arranged opposite to each other at the protein surface in a distance of approximately 30 A, which suggests that they bind into two adjacent major grooves of B-DNA in an anti-parallel manner. A binding pocket is situated at a strategic position in the dimer interface and could possess a regulatory role altering the positions of the DNA-binding helices.

Assessment of bacterial biofilm on stainless steel by hyperspectral fluorescence imaging

In this study, hyperspectral fluorescence imaging techniques were investigated for detection of microbial biofilm on stainless steel plates typically used to manufacture food processing equipment. Stainless steel coupons were immersed in bacterium cultures of nonpathogenic E. coli, Pseudomonas pertucinogena, Erwinia chrysanthemi, and Listeria innocua. Following a 1-week growth in rich medium tryptic soy broth (TSB) and M9 minimal medium with casamino acids (M9C), biofilm formations were evaluated using a recently developed portable hyperspectral fluorescence imaging system. Hyperspectral fluorescence images of the biofilm samples, in response to ultraviolet-A (320 to 400 nm) excitation, were acquired from 416 to 700 nm. Fluorescence images in the blue emission peak region exhibited the most contrast between biofilms and stainless steel coupons. On the basis of correlation analyses, two-band ratios compared with the single-band images enhanced the contrast between the biofilm forming area and untreated coupon surfaces. A two-band fluorescence ratio image, 444/588 nm, resulted in the greatest contrast between the biofilm formations and stainless steel coupon for the biofilms grown in M9C medium. TSB medium showed relatively high auto-fluorescence, and thus further investigation is needed to mitigate the contribution of strong TSB auto-fluorescence in detection of biofilms.

Long-Term Survival ofEscherichia coliLacking the HipBA Toxin-Antitoxin System during Prolonged Cultivation

We designed and constructed six major toxin-antitoxin disruptants (DeltachpBIK, DeltadinJ-yafQ, DeltahipBA, DeltamazEF, DeltarelBE, and DeltayefM-yoeB) of Escherichia coli K-12 W3110. On prolonged cultivation of these disruptants with minimal M9 medium, the DeltahipBA cells exhibited a significantly longer life span than that of the other disruptants and of wild-type cells, as analyzed with a LIVE/DEAD BacLight kit (Invitrogen, Carlsbad, CA) in combination with flow cytometry analysis. The gene expression level of hipA in the wild-type cells was highest at the stationary phase of 40 h. The DeltahipBA cells showed higher macromolecular synthesis activity than the wild-type cells at the stationary phase. Stationary phase cells of DeltahipBA and the wild-type strain showed a significantly extended life span under anaerobic conditions. Furthermore, the DeltahipBA cells showed higher resistance to H(2)O(2) than the wild type. These results suggest that HipBA induces cell death with oxidative stress during prolonged cultivation. This is the first report that an E. coli toxin-antitoxin (TA) system affects frequency of survival during the long-term stationary phase.

Stable and high energy generation by a strain of Bacillus subtilis in a microbial fuel cell

In this study, the Gram-positive aerobic bacterium Bacillus subtilis has for the first time been employed in a microbial fuel cell (MFC). A glucose-fed MFC with M9 minimal medium in the anode chamber was operated for 3 months, establishing a highly active MFC using filtered M9 medium as the catholyte, carbon cloth as the anode and a 20% platinum electrode as the cathode. The bioelectrical responses of the MFC were characterized by the circuit potential, measured at an average value of 370 mV. A potential of 115 mV appeared to characterize the maximum power produced from a polarization test was 1.05 mW cm −2 at a resistance of 0.56 kΩ. In situ cyclic voltammograms with and without biofilm anodes were performed in the growth phase and showed that redox metabolites were produced, which varied with physiological status. Voltammograms obtained from a comparative study of broth, supernatant and resuspended bacterial cells revealed that the electrochemical activity in the anode chamber arose from the redox compounds in the supernatant. The results show that the microorganism B. subtilis is electrochemically active and that the electron transfer mechanism is mainly due to the excreted redox compounds (mediator) in the broth solution and not to the membrane-bound proteins.

The Klebsiella pneumoniae O2a Antigen Defines a Second Mechanism for O Antigen ATP-binding Cassette Transporters

An ATP-binding cassette (ABC) transporter-dependent mechanism is responsible for the biosynthesis of polysaccharide O antigens in the lipopolysaccharides of many Gram-negative bacteria. In the Escherichia coli O9a prototype, addition of a non-reducing terminal modification regulates chain length. The terminal residue is an export signal recognized by the nucleotide-binding domain component of the cognate ABC transporter. The Klebsiella pneumoniae O2a antigen lacks a capping residue, and the corresponding nucleotide-binding protein does not contain a separate substrate-binding domain. Unlike the O9a transporter, the O2a transporter can export heterologous O antigens. Export of the O2a antigen is obligatorily coupled to chain elongation, and the O2a transporter is essential for establishing O antigen chain length. The E. coli O9a transporter operates after chain length has been determined. Furthermore biosynthesis and export can be uncoupled. O antigen chain length is a critical element in the ability of lipopolysaccharides to confer resistance to complement-mediated killing. This study establishes that two distinctly different approaches are available for the regulation of O antigen chain length and export via an ABC transporter.

An intact SAM‐dependent methyltransferase fold is encoded by the human endothelin‐converting enzyme‐2 gene

A recent survey of protein expression patterns in patients with Alzheimer's disease (AD) has identified ece2 (chromosome: 3; Locations: 3q27.1) as the most significantly downregulated gene within the tested group. ece2 encodes endothelin-converting enzyme ECE2, a metalloprotease with a role in neuropeptide processing. Deficiency in the highly homologous ECE1 has earlier been linked to increased levels of AD-related {beta}-amyloid peptide in mice, consistent with a role for ECE in the degradation of that peptide. Initially, ECE2 was presumed to resemble ECE1, in that it comprises a single transmembrane region of {approx}20 residues flanked by a small amino-terminal cytosolic segment and a carboxy-terminal lumenar peptidase domain. The carboxy-terminal domain has significant sequence similarity to both neutral endopeptidase, for which an X-ray structure has been determined, and Kell blood group protein. After their initial discovery, multiple isoforms of ECE1 and ECE2 were discovered, generated by alternative splicing of multiple exons. The originally described ece2 transcript, RefSeq NM{_}174046, contains the amino-terminal cytosolic portion followed by the transmembrane region and peptidase domain (Fig. 1, isoform B). Another ece2 transcript, available from the Mammalian Gene Collection under MGC2408 (Fig. 1, isoform C), RefSeq accession NM{_}032331, is predicted to be translated into a 255 residue peptidemore » with low but detectable sequence similarity to known S-adenosyl-L-methionine (SAM)-dependent methyltransferases (SAM-MTs), such as the hypothetical protein TT1324 from Thermus thermophilis, PDB code 2GS9, which shares 30% amino acid sequence identity with ECE2 over 138 residues of the sequence. Intriguingly, another 'elongated' ece2 transcript (Fig. 1, isoform A) (RefSeq NM{_}014693) contains an amino-terminal portion of the putative SAM-MT domain, the transmembrane domain, and the protease domain. This suggests the possibility for coexistence of the putative SAM-MT and protease domains in a single polypeptide and their transmembrane interplay. Although sequence conservation across the SAM-MT family is weak, the structural fold is highly conserved. The most conserved part of this fold is the SAM-binding subdomain, which is shared between MGC2408 and hypothetical protein TT1324. Typically, the SAM-binding subdomain is flanked by a variable Nterminal extension and, at the C-terminus, by a substrate- binding subdomain, which varies enormously in size but preserves a conserved topology with three antiparallel b-strands. The 'elongated' transcript of ece2 lacks this substrate-binding subdomain. To test the hypothesis that the 255 residue ece2 gene product MGC2408 represents a complete SAM-MT fold, we have determined a crystal structure of this protein in the presence of SAH.« less

Carbohydrate Binding Mechanism of the Macrophage Galactose-type C-type Lectin 1 Revealed by Saturation Transfer Experiments

Macrophage galactose-type C-type lectins 1 and 2 (MGL1/2) are expressed on the surfaces of macrophages and immature dendritic cells. Despite the high similarity between the primary sequences of MGL1 and MGL2, they display different ligand specificities. MGL1 shows high affinity for the LewisX trisaccharide, whereas MGL2 shows affinity for N-acetylgalactosamine. To elucidate the structural basis for the ligand specificities of the MGLs, we performed NMR analyses of the MGL1-LewisX complex. To identify the LewisX binding site on MGL1, a saturation transfer experiment for the MGL1-LewisX complex where sugar-CH/CH2-selective saturation was applied was carried out. To obtain sugar moiety-specific information on the interface between MGL1 and the LewisX trisaccharide, saturation transfer experiments where each of galactose-H5-, fucose-CH3-, and N-acetylglucosamine-CH3-selective saturations was applied to the MGL1-LewisX complex were performed. Based on these results, we present a LewisX binding mode on MGL1 where the galactose moiety is bound to the primary sugar binding site, including Asp-94, Trp-96, and Asp-118, and the fucose moiety interacts with the secondary sugar binding site, including Ala-89 and Thr-111. Ala-89 and Thr-111 in MGL1 are replaced with arginine and serine in MGL2, respectively. The hydrophobic environment formed by a small side chain of Ala-89 and a methyl group of Thr-111 is a requisite for the accommodation of the fucose moiety of the LewisX trisaccharide within the sugar binding site of MGL1.

Structural Analysis of a Periplasmic Binding Protein in the Tripartite ATP-independent Transporter Family Reveals a Tetrameric Assembly That May Have a Role in Ligand Transport

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 A resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 A root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 A) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides alpha-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.

Aerobic Fermentation of d -Glucose by an Evolved Cytochrome Oxidase-Deficient Escherichia coli Strain

Fermentation of glucose to D-lactic acid under aerobic growth conditions by an evolved Escherichia coli mutant deficient in three terminal oxidases is reported in this work. Cytochrome oxidases (cydAB, cyoABCD, and cbdAB) were removed from the E. coli K12 MG1655 genome, resulting in the ECOM3 (E. coli cytochrome oxidase mutant) strain. Removal of cytochrome oxidases reduced the oxygen uptake rate of the knockout strain by nearly 85%. Moreover, the knockout strain was initially incapable of growing on M9 minimal medium. After the ECOM3 strain was subjected to adaptive evolution on glucose M9 medium for 60 days, a growth rate equivalent to that of anaerobic wild-type E. coli was achieved. Our findings demonstrate that three independently adaptively evolved ECOM3 populations acquired different phenotypes: one produced lactate as a sole fermentation product, while the other two strains exhibited a mixed-acid fermentation under oxic growth conditions with lactate remaining as the major product. The homofermenting strain showed a D-lactate yield of 0.8 g/g from glucose. Gene expression and in silico model-based analyses were employed to identify perturbed pathways and explain phenotypic behavior. Significant upregulation of ygiN and sodAB explains the remaining oxygen uptake that was observed in evolved ECOM3 strains. E. coli strains produced in this study showed the ability to produce lactate as a fermentation product from glucose and to undergo mixed-acid fermentation during aerobic growth.

Serum Inhibits P. aeruginosa Biofilm Formation on Plastic Surfaces and Intravenous Catheters

Biofilm formation on medical devices such as intravenous catheters is a serious manifestation of Pseudomonas aeruginosa infections. Serum has bactericidal activity, a function of multiple serum components. In this study, we determined the effect of serum and serum components on the formation of P. aeruginosa biofilm. We examined the effect of adult bovine serum (ABS) or bovine serum albumin (BSA) on biofilm development on plastic coverslips. This was done using both static and continuous flow-through culture systems and P. aeruginosa strain PAO1. Biofilms were quantified using crystal violet assays and visualized using confocal scanning laser microscopy and scanning electron microscopy. We examined the effect of ABS on PAO1 swimming and twitching motilities (both contribute to P. aeruginosa biofilm development). We also analyzed the inhibitory effect of adult human serum (AHS) and plasma (AHP) on PAO1 biofilm development on plastic coverslips and intravenous catheters. Compared with M9 minimal medium (M9), 10% ABS-supplemented medium (M9/ABS-10) caused a significant decrease in biofilm development. Coverslips precoated with M9/ABS-10 failed to develop biofilm when placed in M9. In addition to reduced biofilm formation, adding ABS to M9 reduced an already-developed PAO1 biofilm. Compared with M9, M9/ABS-10 enhanced PAO1 twitching motility considerably, but did not affect swimming motility. Similar to ABS, BSA blocked biofilm formation but did not affect PAO1 twitching motility. Both AHS and AHP blocked PAO1 biofilm formation on plastic coverslips and intravenous catheters. These results suggest that as part of the host innate resistance, serum inhibits P. aeruginosa biofilm formation on plastic surfaces, including intravenous catheters. Two possible scenarios for this inhibition include blocking the direct interaction between P. aeruginosa and the substrates, and the enhancing P. aeruginosa twitching motility.

The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways

There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N- and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.

Natural Homolog of tRNA Synthetase Editing Domain Rescues Conditional Lethality Caused by Mistranslation

AlaXp is a widely distributed (from bacteria to humans) genome-encoded homolog of the editing domain of alanyl-tRNA synthetases. Editing repairs the confusion of serine and glycine for alanine through clearance of mischarged (with Ser or Gly) tRNA(Ala). Because genome-encoded fragments of editing domains of other synthetases are scarce, the AlaXp redundancy of the editing domain of alanyl-tRNA synthetase is thought to reflect an unusual sensitivity of cells to mistranslation at codons for Ala. Indeed, a small defect in the editing activity of alanyl-tRNA synthetase is causally linked to neurodegeneration in the mouse. Although limited earlier studies demonstrated that AlaXp deacylated mischarged tRNA(Ala) in vitro, the significance of this activity in vivo has not been clear. Here we describe a bacterial system specifically designed to investigate activity of AlaXp in vivo. Serine toxicity, experienced by a strain harboring an editing-defective alanyl-tRNA synthetase, was rescued by an AlaXp-encoding transgene. Rescue was dependent on amino acid residues in AlaXp that are needed for its in vitro catalytic activity. Thus, the editing activity per se of AlaXp was essential for suppressing mistranslation. The results support the idea that the unique widespread distribution of AlaXp arises from the singular difficulties, for translation, poised by alanine.

High-titer production of monomeric hydroxyvalerates from levulinic acid in Pseudomonas putida

Hydroxyacids represent an important class of compounds that see application in the production of polyesters, biodegradable plastics and antibiotics, and that serve as useful chiral synthetic building blocks for other fine chemicals and pharmaceuticals. An economical, high-titer method for the production of 4-hydroxyvalerate (4HV) and 3-hydroxyvalerate (3HV) from the inexpensive and renewable carbon source levulinic acid was developed. These hydroxyvalerates were produced by periodically feeding levulinate to Pseudomonas putida KT2440 expressing a recombinant thioesterase II ( tesB) gene from Escherichia coli K12. The titer of 4HV in shake flask culture reached 13.9 ± 1.2 g L −1 from P. putida tesB + cultured at 32 °C in LB medium periodically supplemented with glucose and levulinate. The highest 3HV titer obtained was 5.3 ± 0.1 g L −1 in M9 minimal medium supplemented with glucose and levulinate.

High-level expression and purification of the second transmembrane domain of wild-type and mutant human melanocortin-4 receptor for solid-state NMR structural studies

It has been demonstrated that human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and heterozygous mutations in the hMC4R gene are the most frequent genetic cause of severe human obesity. In order to obtain additional insight into the structure and function, we cloned, expressed, and purified the second transmembrane domain of the wild-type hMC4R (wt-TM2) and D90N mutant hMC4R (m-TM2). To facilitate structural studies of these hMC4R by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled protein are necessary. However, large-scale production of most transmembrane proteins has been limited by experimental adversities due to insufficient yields and low solubility of protein. Nevertheless, through the optimization of the expression and purification approach, we could obtain uniformly or selectively labeled fusion proteins in yields as high as 200–250 mg per liter M9 minimal medium. These proteins were overexpressed in inclusion bodies as a fusion protein with ketosteroid isomerase (KSI) in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography under denaturing conditions. wt-/m-TM2 peptides were released from the fusion by cyanogen bromide cleavage at the Met residue and separated from the carrier KSI by size exclusion chromatography. Initial structural data obtained by solution NMR measurements of wt-/m-TM2 is also presented. The successful application to the production of the second transmembrane domain of human MC4R indicates that the method can be applied to other transmembrane proteins as well and also enable its structural and functional studies using solid-state NMR spectroscopy.