Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Alignment of protein sequences suggests that this protein belongs to the AbgT family of transporters19. Mapping of the mini-Tn5 insertion in the mutant Aj17 of A. jadensis revealed disruption of a gene which putatively encodes a DNA repair system specific for alkylated DNA exhibiting 61% identical amino acids to the homologous gene of A. borkumensis SK2. (Fig.3C).3C). . Chernikova TN, Bargiela R, Toshchakov SV, Shivaraman V, Lunev EA, Yakimov MM, Thomas DN, Golyshin PN. provided by wikipedia EN. Its ubiquity, unusual physiology and demonstrated role in biodegradation show that it is globally important . Hyde JE, Dittrich S, Wang P, Sims PFG, de Crcy-Lagard V, Hanson AD. Growth and fatty acid content of A. borkumensis SK2 mini-Tn5-induced mutants and knockout mutants defective in lipid export growing on pyruvate as sole carbon source. The accumulation of lipophilic substances in bacteria is normally accompanied by morphological changes of the cells. Sodium ion enhances sulfamethazine efflux via YdaH. The purified protein was then concentrated to 20 mg/ml in a buffer containing 20 mM Na-HEPES (pH 7.5) and 0.05% DDM. We then used valence calculations24 to predict if this location can form a metal ion-binding site. The membrane protein was then solubilized in 2% (w/v) n-dodecyl--D-maltoside (DDM). Further analyses also suggest that YdaH could act as an antibiotic efflux pump and mediate bacterial resistance to sulfonamide antimetabolite drugs. At present, the influence of some transport proteins on the export of neutral lipids is being investigated in our laboratory. Plasmid DNA was isolated using the method described by Birnboim and Doly (9). The initial identification of enzymes involved in folate biosynthesis was largely carried out in the 196070s, leading to the detailed description of this enzymatic pathway1116. The https:// ensures that you are connecting to the The ydaH gene of Alcanivorax borkumensis encodes an integral membrane protein of the AbgT family of transporters for which no structural information was available. (Fig.3G3G). The site is secure. IX Purification and properties of the enzymes required for the formation of dihydropteroic acid. We also found that the minimum inhibitory concentrations (MICs) of BL21(DE3)abgTpabA/pET15bydaH cells to sulfadiazine and sulfathiazole were both greater than eight times than those of BL21(DE3)abgTpabA cells carrying the empty vector pET15b. This protein belongs to a three-component Czc (Cd2+, Zn2+, and Co2+) chemiosmotic efflux pump of soil microorganisms (40). The global feature of the completely sequenced Alcanivorax borkumensis SK2 type strain chromosome is its symmetry and homogeneity. FOIA Nas, Na+ symporter family protein; hyp, hypothetical protein; tsp, two-component sensor protein; tr, transcription regulator, putative; rr, response regulator; shk, sensor histidine kinase; RND, probable RND efflux transporter (mutant ABO_27/56). However, among proteins in this diverse family, only E. coli AbgT17,18 and N. gonorrhoeae MtrF20,21 have been partially characterized. Mapping of the mini-Tn5 insertion in the genome of the A. borkumensis mutant ABO_27/56 revealed a Na+ symporter family protein and an RND efflux transporter adjacent to the site of mini-Tn5 insertion (Fig. In contrast to the two Alcanivorax strains, cells of A. baylyi strain ADP1 accumulated TAGs and WEs exclusively intracellularly as insoluble inclusions under growth-limiting conditions. The biosynthesis of folic acid: II. To confirm that the presence of Na+ enhances the function of the YdaH transporter, we next measured the efflux of radioactive sulfamethazine that had accumulated in strain BL21(DE3)abgTpabA/pET15bydaH, both in the absence and presence of Na+ or K+. For the Na+-dependent experiments, cells were incubated with 75 nM [3H]-sulfamethazine in the presence of NaCl (0, 5 or 100 mM) for 15 min, then applied directly to prewetted glass-fiber filters, and washed immediately twice with 5-ml aliquots of buffer containing 100 mM Tris-HCl (pH 7.5) and 0, 5 or 100 mM NaCl. Crystals of the tungsten cluster derivative were prepared by incubating the crystals of YdaH in solution containing 32% PEG 400, 0.1 M sodium acetate (pH 5.0), 0.05 M magnesium acetate, 3% glycerol and 2% (w/v) OG, 0.05% (w/v) DDM and 0.5 mM Na9[-SiW9O34H] 23H2O for 4 hours at 25C. Whereas cells of A. borkumensis SK2 showed strong fluorescence already after 3 days of incubation, cells of A. jadensis T9 and A. baylyi strain ADP1 showed only a weak fluorescence. Again, these data indicate that residues D180, N390, W400, P418, R426, D429 and N433 may be critical for the function of the A. borkumensis YdaH transporter. A. borkumensis was grown on ONR7a medium at 30C and harvested in the stationary growth phase by centrifugation at 4,000 rpm for 50 min and 4C. No significant presence of extracellular TAGs was observed in culture supernatants of either transconjugants or knockout mutants. Federal government websites often end in .gov or .mil. Carter EL, Jager L, Gardner L, Hall CC, Willis S, Green JM. Tseng TT, Gratwick KS, Kollman J, Park D, Nies DH, Goffeau A, Saier MH., Jr The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development protein. Based on additional experimental data, we propose that YdaH could function as a drug efflux pump, capable of removing sulfonamide antimetabolites and mediate resistance to this class of drugs in bacterial cells. The use of lipophilic stains, such as NR and SB38, allowed the rapid identification and selection of transconjugants showing defects in lipid metabolism. A. borkumensis is found naturally in seawater environments. Alcanivorax balearicus Rivas et al. Thus, loss of the PMF may inactivate pump activity, which results in in enhanced accumulation of substrates. Ultrathin sections of cells were analyzed by TEM as reported previously (44). The origin and terminus of replication are located opposite to each other in the chromosome and are discerned with high signal to noise ratios by maximal oligonucleotide usage biases on the leading and lagging strand. includes: . Based on the obtained sequences, primers were designed to map the entire region of the insertion site in the mutant of A. jadensis. Alcanivorax borkumensis can be found in every ocean, drifting with the current. Reynolds JJ, Brown GM. Viewed in parallel to the membrane, the dimer is about 60 tall, 80 wide and 60 thick, with the transmembrane portion of the transporter lying approximately in the middle (Fig. Olivera NL, Nievas ML, Lozada M, Del Prado G, Dionisi HM, Sieriz F. Res Microbiol. Why and how cells of Alcanivorax species export neutral lipids is unknown. Bethesda, MD 20894, Web Policies Extracellular lipids were extracted directly from the supernatant fraction (corresponding to the amount of dry cell material to analyze), which was mixed with the same volume of chloroform-methanol (2:1 [vol/vol]) and vortexed. However, the level of intracellular folic acid concentration in cells expressing R426A was nearly identical to that of the double knockout strain carrying wild-type YdaH (Fig. Each protomer of YdaH in the dimer contains nine transmembrane helices (TMs 19 and TMs 1-9, respectively) and two helical hairpins (HPs 12 and HPs 1-2, respectively) (Fig. 2015; Alcanivorax hongdengensis Wu et al. The new PMC design is here! Crystals of YdaH belonged to space group P21 (Table 1). It is found in low numbers in all oceans of the world and becomes abundant in oil-contaminated waters. For A. borkumensis, the gene products are tnpB, transposon B protein; czcA2, heavy metal RND efflux transporter, CzcA family; czcB2, heavy metal RND efflux membrane fusion protein, CzcB family; and czcC2, heavy metal RND efflux outer membrane protein, CzcC family (mutant ABO_6/39). The .gov means its official. These transconjugants showed reduced fluorescence under UV light or were phenotypically different in terms of the border surrounding the streaks compared to the wild-type strain. Mutations in dihydropteroate synthase are responsible for sulfon and sulfonamide resistance in, Brown GM, Weisman RA, Molnar DA. Brown GM. Here, we report the crystal structure of A. borkumensis YdaH, revealing a dimeric molecule with an architecture distinct from other families of transporters. This basin probably allows aqueous solution to reach the midpoint of the membrane bilayer. A 2 l protein solution containing 20 mg/ml YdaH in 20 mM Na-HEPES (pH 7.5) and 0.05% (w/v) DDM was mixed with a 2 l of reservoir solution containing 28% PEG 400, 0.1 M sodium acetate (pH 5.0), 0.05 M magnesium acetate, 3% glycerol and 2% (w/v) n-octyl--D-glucoside (OG). The potential of the folic acid biosynthesis pathway as a target for the development of antibiotics has been clinically validated. (Fig.2).2). The transconjugants ABO_26/1 and ABO_19/48 grew to cell densities of 0.5 and 0.6 g/liter, with intracellular lipid contents between 2.75 and 4.56%, respectively, while transconjugant ABO_25/21 grew to a cell density of 0.7 g/liter and accumulated 4.89% of lipids. Samples were taken after 12 h (early exponential growth phase), 48 h (late exponential growth phase), and 96 h (stationary growth phase) for A. borkumensis SK2 and after 32 h (exponential growth phase) and 60 h and 72 h (stationary growth phase) for A. jadensis T9. Coordinating with this bound Na+ ion are the side chains of N390, D429, N433 and the backbone carbonyl oxygens of G394 and D429 (Fig. How they work The species breaks down crude oil using a range of enzymes it produces naturally. All cells, both prokaryotic and eukaryotic, require folic acid for the biosynthesis of a diverse range of cellular components. The biosynthesis of folic acid. It is interesting to note that sulfonamides were used between the late 1930s and early 1940s to treat gonorrhea, but the rapid emergence of strains resistant to this class of drug has resulted in its removal from the list of recommended drug therapies27. Crystal structures of the CusA heavy-metal efflux pump suggest methionine-mediated metal transport mechanism. To confirm the phenotype of the transconjugants defective in lipid export, the genes algL (mutants ABO_10/30 and ABO_19/48), algE (transconjugant ABO_26/1), and MATE (transconjugant ABO_25/21) were also disrupted by insertion of an Sm resistance gene cassette. Inhibition by sulfonamides. Alginate plays a key role as a virulence factor of plant-pathogenic pseudomonads in the formation of biofilms and with the encystment process of Azotobacter spp. The bacterium multiplies at a rapid rate in areas with high concentrations of oil compounds. Because of this finding, it is hypothesized that AbgT-family transporters19 contribute to the bacterial folate synthesis pathway by importing p-aminobenzoyl-glutamate for producing this essential vitamin. The dimeric form of YdaH in the crystal lattice is also in good agreement with the oligomerization state of this membrane protein in solution where it assembles as a dimer (Supplementary Fig. Hexadecane applied on a filter paper in the lid was used as carbon and energy source. All oligonucleotides were purchased from (Integrated DNA Technologies, Inc., Coralville, IA) in a salt-free grade. The presence of Na+ significantly enhances [3H]-sulfamethazine efflux in BL21(DE3)abgTpabA/pET15bydaH cells (black, control cells with the empty vector; red, 0 mM NaCl; magenta, 5 mM KCl; blue, 5 mM NaCl). Ultrathin sections of Alcanivorax strain cells were analyzed as described previously (32). In this study, pilot-scale Alcanivorax borkumensis fermentation was developed for producing large volumes of petroleum degrading enzymes cocktail . Cells transformed with pET15bydaH or pET15b were grown in liquid minimal medium supplemented with 30 nM PABA and harvested when the optical density (OD600 nm) reached 0.5. 436E, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne. No differences in the survivals of cells could be observed between A. borkumensis SK2 and a knockout mutant with reduced storage lipid accumulation (32). The adjacent region contains a zinc ABC-transport uptake system. A list of our current non-bibliographic LinkOut providers can be found here. Error bars denote standard deviation (n = 3). However, the cell form changed slightly, as was obvious from the TEM. When the OD600 nm reached 0.4, 100 mg/l of lysine, phenylalanine and threonine, 50 mg/l isoleucine, leucine and valine, and 60 mg/l of L-selenomethionine were added. Marine hydrocarbonoclastic bacteria, like Alcanivorax borkumensis, play a globally important role in bioremediation of petroleum oil contamination in marine ecosystems.Accumulation of storage lipids, serving as endogenous carbon and energy sources during starvation periods, might be a potential adaptation mechanism for coping with nutrient limitation, which is a frequent stress factor . http://www.carolina.com/catalog/search- SearchForm. Its ubiquity, unusual physiology and demonstrated role in biodegradation show that it is globally important . In the same experiment, Chronopoulou et al. The A disruption in the genome of the A. borkumensis mutant ABO_27/29 was localized in a gene encoding a sodium dicarboxylate symporter family. Iterations of refinement using PHENIX41 and CNS42 and model building in Coot39 lead to the 4.10 -resolution structural model of the YdaH transporter. Site-directed point mutations on residues D180, N390, W400, P418, R426, D429 and N433, which are expected to be critical for the function of the YdaH transporter, were performed to generate the single point mutants D180A, N390A, W400A, P418A, R426A, D429A and N433A. 6 and Supplementary Table 3). How Are Antibodies Used for Blood Typing? I know you can buy it from ATCC but it is very expensive. [Regularities of the location of genes having different functions and of some other nucleotide sequences in the bacterial chromosome]. Cells were grown in Luria Broth (LB) medium with 100 g/ml ampicillin at 37C. The biosynthesis of folic acid: I. Substrate and cofactor requirements for enzymatic synthesis by cell-free extracts of. The specific activity of [3H]-PABA is 30.4 Ci/mmol. Substructure solution with SHELXD. Here we show that YdaH is capable of exporting the folate metabolite PABA from bacterial cells. Gene organization of the loci identified by mini-Tn5 mutagenesis of Alcanivorax strains. YdaH is a bowl-shaped . Cryoprotection was achieved by raising the PEG 400 concentration to 32% in a single step. (54) and with an ABI Prism 3730 capillary sequencer at the Universittsklinikum Mnster (UKM). (Fig.4B4B). Insoluble material was removed by ultracentrifugation at 100,000 x g. The extracted protein was purified with a Ni2+-affinity column. However, many pathogens have developed resistance to these antibiotics, prompting a reevaluation of potential drug targets within the pathway. Ethayathulla AS, Yousef MS, Amin A, Leblanc G, Kaback HR, Guan L. Structure-based mechanism for Na. Of the nine TMs, four (TM2, TM3, TM7 and TM8) are broken into segments within the membrane. 1998, 2706794854: Alcanivorax borkumensis JGI ETNP_300m_187_B10 (contamination screened), Alcanivorax borkumensis Yakimov, Golyshin, Lang, Moore, Abraham, Lunsdorf & Timmis, 1998. A water molecule also participates in the interaction, forming another coordinate bond with the Na+ ion. The CzcABC complex is an efflux pump that functions as a chemiosmotic divalent cation/proton antiporter (41). A. borkumensis SK2 grew to a cell density of about 0.8 g/liter, intracellularly accumulating TAGs that were about 5% of its cell dry weight (CDW) when cultivated with pyruvate as the sole carbon source. Database (Oxford). Hybrid cosmids of the resulting clones harbored several PstI fragments, including one which contains the mini-Tn5 and adjacent genomic DNA to the mini-Tn5 insertion locus. The resulting phases were of excellent quality, which enabled us to trace of most of the molecule. A. borkumensis shares a similar oligonucleotide usage and promoter structure with the Pseudomonadales. Biofilms that floated from the surface were dominated by obligate hydrocarbonoclastic bacteria, with Alcanivorax borkumensis constituting almost half of the total bacterial community. Alginate biosynthesis is a well-characterized process in species of Pseudomonas and Azotobacter. Based on the export of lipids by Alcanivorax strains, a screening method was developed to select transconjugants defective in biosynthesis or export of neutral lipids. Cells were grown in 12 l of LB medium with 100 g/ml ampicillin at 25C. We then expressed the YdaH transporter in this double knockout strain using the plasmid pET15bydaH. This study was supported by a grant provided by the Deutsche Forschungsgemeinschaft to A.S. (Ste386/7-3). Reproduction of material from this website without written permission is strictly prohibited. In addition, accumulation of TAGs and WEs was promoted when the cells were cultivated under nitrogen-limited conditions (data not shown). The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is able to degrade mixtures of n-alkanes as they occur in marine oil spills.However, investigations of growth behavior and physiology of these bacteria when cultivated with n-alkanes of different chain lengths (C 6 to C 30) as the substrates are still lacking.Growth rates increased with increasing alkane chain length up to a . Here, we investigate the effect of solution and interfacial conditions on the growth of A. borkumensis. Lipid extracts obtained from 5 mg lyophilized cells (C) or from the corresponding amount of supernatant (S) from cells grown on different carbon sources were applied per lane. A. 2007; Alcanivorax borkumensis Yakimov et al. (a) The inner core of YdaH, comprising TMs 1, 2, 5, 6 and 7 (colored slate), contributes to dimerization as well as formation of a frame-like structure housing the outer core of the protomer. Ten microliter injections of the ligand solution were used for data collection. Extracts obtained from 3 mg cells (A) (intracellular) and the corresponding amount of supernatants (B) (extracellular) were analyzed. Received 2009 May 29; Accepted 2009 Nov 6. In addition, the presence of a border was more evident in cells growing on hexadecane than in cells growing on pyruvate, which could be related to the export of neutral lipids by A. jadensis. However, cells expressing R426A are able to decrease the [3H]-PABA concentration when compared with cells carrying wild-type YdaH. That CCCP appears to inhibit the pump activity, indicating that YdaH is PMF-dependent. HHS Vulnerability Disclosure, Help These results demonstrated that a combination of NR and SB38 may be used to detect mutants defective in lipid biosynthesis and export. Substances were identified by comparison of their retention times with those of authentic standard fatty acid methyl esters. When transformed with plasmid expressing the mutant transporter, D180A, N390, W400A, P418A, D429A and N433A, folic acid production was significantly increased in these cells. A comparatively low number of only 18 genome islands with atypical oligonucleotide usage was detected in the A. borkumensis chromosome. The kanamycin resistant gene was then released to generate the BL21(DE3)abgT knockout strain. Careers. Specifically, we determined the accumulation level of radioactive sulfamethazine in strain BL21(DE3)abgTpabA/pET15bydaH in the presence of 5 or 100 mM NaCl. It can consume a wider range of . We then measured the accumulation of [3H]-PABA in cells expressing these mutant transporters. 5). 3A and B). Based on the values of KA, the change in free energy (G) and entropy (S) were calculated with the equation: G = RT lnKA = H TS, where T is 273 K and R is 1.9872 cal/K per mol. As PABA is an important precursor for producing essential folic acid in bacteria, we did not understand why the function of YdaH appeared to lower the intracellular PABA concentration. (1998) 48:339-348. Lipid inclusions in both Alcanivorax strains showed extreme heterogeneity in shape and size, comprising spherical, rectangular, disc-shaped, and irregularly shaped inclusions. The resultant mixture was equilibrated against 500 l of the reservoir solution at 25C. A. jadensis was grown in MWM medium at 30C until reaching the stationary phase (about 72 h). Our data suggest that A. borkumensis YdaH could function as an antibiotic efflux pump and remove sulfonamides from the cell to mediate bacterial resistance to this class of antimetabolites. Lipids from supernatant samples were extracted directly from the culture broth with chloroform-methanol (1:1 [vol/vol]). 1.Cells of A. borkumensis were cultivated aerobically at 30C and 150 rpm for 60 h in ONR7a medium containing 1% (wt/vol) sodium pyruvate or 0.5% (wt/vol) hexadecane as the sole carbon source.Cells of A. jadensis were cultivated at 30C and 150 rpm for 96 h in . After tracing the initial model manually using the program Coot39, the model was refined using PHENIX41 leaving 5% of reflections in Free-R set. NCBI Taxonomy: a comprehensive update on curation, resources and tools. Brown MH, Paulsen IT, Skurray RA. Alginate lyases have been isolated from a variety of marine bacteria, soil bacteria, and fungi, and in some cases, such as for certain Pseudomonas spp., the presence of more than one alginate lyase has been reported (67). Members of at least three types of transports systems, the ABC-type, RND-type, and MFS-type transporters, have been proposed to function together with MFP to facilitate transport across both membranes of the cell envelope of gram-negative bacteria (18, 20, 42, 52). Residues D180, N390, W400, P418, R426, D429 and N433 are in green sticks. You may print and distribute up to 200 copies of this document annually, at no charge, for personal and classroom educational use. The biosynthesis of folic acid. Crystal Structures of CusC Review Conformational Changes Accompanying Folding and Transmembrane Channel Formation. To support the drug susceptibility testing results, we measured the accumulation of radioactive sulfamethazine in BL21(DE3)abgTpabA cells that carried pET15bydaH or pET15b. Alcanivorax borkumensis is a hydrocarbon degrading bacterium linked to oil degradation around oil spill sites. Alcanivorax borkumensis (A. borkumensis) is a Gram-negative, rod-shaped hydrocarbonoclastic ("oil-degrading") bacterium. They were therefore analyzed for their capability to synthesize and export lipids. Based on the crystal structure, we found that residues N390, D429, N433 are involved in coordinating the bound Na+ ion. 8600 Rockville Pike Use of glycerol-crypotected, Unemo M, Shafer WM. Because of the rapid adaption of bacterial pathogens to antimicrobials, drug resistance to currently administered antibiotics is a serious problem that is rising at an alarming rate. 2a). 2012 Jul 16;586(15):2043-8. doi: 10.1016/j.febslet.2012.03.051. Schneider TR, Sheldrick GM. Here, we report the crystal structure of A. borkumensis YdaH, revealing a dimeric molecule with an architecture distinct from other families of transporters. These phases were then improved by NCS averaging with density modification using the program RESOLVE38. 1c and d). When transformed with plasmids expressing the mutant transporters D180A, N390A, W400A, P418A, D429A and N433A, folic acid production was significantly increased in these cells. To observe whether synthesis of lipids by Alcanivorax provokes changes of the cell morphology, we have conducted experiments with both Alcanivorax species grown in the presence of hexadecane. The solvent system hexane-diethyl ether-acetic acid (90:7.5:1 [vol/vol/vol]) was used for elution, and lipids were visualized by staining with iodine vapor. Occurrence of TAGs and WEs by A. jadensis T9 was analyzed using acetate, pyruvate, or hexadecane as the sole carbon and energy source. The ydaH gene of Alcanivorax borkumensis encodes an integral membrane protein of the AbgT family of transporters for which no structural information was available. For quantitative analysis, tridecanoic acid was added to the samples before performing the acidified methanolysis as an internal standard. In lipid bilayer experiments, it has been observed that GDP-mannuronic acid could partially block this pore. An official website of the United States government. They hope that their new understanding of the biochemistry of the micro-organism will lead to the development of new, effective, environmentally friendly methods to clean up oil-contaminated waters. It is possible that other members of the AbgT family of transporters may serve as antimetabolite efflux pumps to protect cells against these noxious agents. (b) Ribbon diagram of a dimer of YdaH viewed in the membrane plane. The corresponding knockout mutants showed growth to cell densities of 0.7 (algE mutant) and 0.6 (algL and MATE mutants) g/liter, with intracellular lipid contents of 3.56, 4.87, and 3.78%, respectively. The morphology of cells growing in the exponential or stationary phase was analyzed by TEM (Fig. Error bars represent standard deviation (n = 3). 6a), suggesting that the uncoupler CCCP was able to inhibit the function of this pump. The problem is of particular severity in treating the parasitic malaria disease. This bacterium is significant . Genomic DNA structure is rather uniform throughout the chromosome with respect to intrinsic curvature, position preference or base stacking energy. About 5,000 mutants of A. borkumensis and 4,000 mutants of A. jadensis were analyzed and screened. The same phenotype was observed with the constructed knockout mutants, in which the presence of extracellular lipids was partially or totally impeded (Table (Table4).4). For identification of the intra- and extracellular DEs produced by A. jadensis T9, lipid extracts from cells and supernatants were isolated from preparative TLC plates and subjected to analysis by ESI/MS2. Niche-specificity factors of a marine oil-degrading bacterium Alcanivorax borkumensis SK2. Do you know anywhere else to acquire it for cheaper than $300. Fatty acid methyl esters were analyzed using an Agilent 6850 GC (Agilent Technologies, Waldbronn, Germany) equipped with a BP21 capillary column (50 m by 0.22 mm; film thickness, 250 nm; SGE, Darmstadt, Germany) and a flame ionization detector (Agilent Technologies, Waldbronn, Germany). Specifically, sulfamethazine was chosen because of the availability of its radioactive analog. Blue dextran (Sigma-Aldrich, St. Louis, MO) was used to determine the column void volume. The https:// ensures that you are connecting to the Wang P, Read M, Sims PFG, Hyde JE. Therefore, we decided to measure the intracellular folic acid concentration microbiologically using Lactobacillus casei26. The strain uses aliphatic hydrocarbons as its main carbon source . Is this connected to that? The fluorescent border was less evident in cells of A. jadensis T9 grown on MWM plates with Nile red and Solvent Blue 38 containing pyruvate (C) than in cells grown with hexadecane as the sole carbon source (D). The observation that cells of A. jadensis showed a border surrounding the streaks and that this border could be related to the amount of lipid produced and/or exported by the cells led to the decision to use agar plates containing NR alone, NR plus SB38, or SB38 alone to identify and select mutants with defects in lipid biosynthesis and/or export. These stains were used in the past to investigate processes involving lipophilic substances (15, 34, 37, 39, 62). The crystallization conditions for SeMet-YdaH were the same as those for the native YdaH protein. Intra- and extracellular lipids of A. jadensis T9 were analyzed by electrospray ionization mass spectrometry (ESI/MS) and ESI/tandem MS (MS2) in the positive-ion mode on a Quattro LCZ (Waters-Micromass, Manchester, United Kingdom) with stating nanospray using capillary with internal wire contact.
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