Microbacterium Liquefaciens Research Articles

The evolution of reduced facilitation in a four-species bacterial community

Abstract Microbial evolution is typically studied in monocultures or in communities of competing species. But microbes do not always compete and how positive inter-species interactions drive evolution is less clear: Initially facilitative communities may either evolve increased mutualism, increased reliance on certain species according to the Black Queen Hypothesis (BQH), or weaker interactions and resource specialization. To distinguish between these outcomes, we evolved four species for 44 weeks either alone or together in a toxic pollutant. These species initially facilitated each other, promoting each other’s survival and pollutant degradation. After evolution, two species (Microbacterium liquefaciens and Ochrobactrum anthropi) that initially relied fully on others to survive continued to do so, with no evidence for increased mutualism. Instead, Agrobacterium tumefaciens and Comamonas testosteroni (Ct) whose ancestors interacted positively, evolved in community to interact more neutrally and grew less well than when they had evolved alone, suggesting that the community limited their adaptation. We detected several gene loss events in Ct when evolving with others, but these events did not increase its reliance on other species, contrary to expectations under the BQH. We hypothesize instead that these gene loss events are a consequence of resource specialization. Finally, co-evolved communities degraded the pollutant worse than their ancestors. Together, our results support the evolution of weakened interactions and resource specialization, similar to what has been observed in competitive communities.

Four new Microbacterium species isolated from seaweeds and reclassification of five Microbacterium species with a proposal of Paramicrobacterium gen. nov. under a genome-based framework of the genus Microbacterium.

The taxonomic relationships of 10 strains isolated from seaweeds collected from two beaches in Republic of Korea were studied by sequencing and analyses of 16S rRNA genes and whole genomes. For the construction of a more reliable and robust 16S rRNA gene phylogeny, the authentic and nearly complete 16S rRNA gene sequences of all the Microbacterium type strains were selected through pairwise comparison of the sequences contained in several public databases including the List of Prokaryotic names with Standing in Nomenclature (LPSN). The clustering of the ten study strains into five distinct groups was apparent in this single gene-based phylogenetic tree. In addition, the 16S rRNA gene sequences of a few type strains were shown to be incorrectly listed in LPSN. An overall phylogenomic clustering of the genus Microbacterium was performed with a total of 113 genomes by core genome analysis. As a result, nine major (≥ three type strains) and eight minor (two type strains) clusters were defined mostly at gene support index of 92 and mean intra-cluster OrthoANIu of >80.00%. All of the study strains were assigned to a Microbacterium liquefaciens clade and distributed further into four subclusters in the core genome-based phylogenetic tree. In vitro phenotypic assays for physiological, biochemical, and chemotaxonomic characteristics were also carried out with the ten study strains and seven closely related type strains. Comparison of the overall genomic relatedness indices (OGRI) including OrthoANIu and digital DNA-DNA hybridization supported that the study strains constituted four new species of the genus Microbacterium. In addition, some Microbacterium type strains were reclassified as members of preexisting species. Moreover, some of them were embedded in a new genus of the family Microbacteriaceae based on their distinct separation in the core genome-based phylogenetic tree and amino acid identity matrices. Based on the results here, four new species, namely, Microbacterium aurugineum sp. nov., Microbacterium croceum sp. nov., Microbacterium galbinum sp. nov., and Microbacterium sufflavum sp. nov., are described, along with the proposal of Paramicrobacterium gen. nov. containing five reclassified Microbacterium species from the "Microbacterium agarici clade", with Paramicrobacterium agarici gen. nov., comb. nov. as the type species.

Platelet Storage: Time to Rethink the Cold

Platelet concentrates (PCs) are widely used in transfusion medicine for therapeutic purposes and their demand is constantly on the rise. Current storage regulations make this product highly susceptible to bacterial contaminations and platelet storage lesions (PSL) causing the need for alternative storage methods to be considered. The implementation of cold storage not only reduces unnecessary wastage of valuable donations and overall costs but also decreases both the risk of bacterial contamination and the occurrence of PSL. The current study aimed at determining how a prolonged cold storage may affect PCs. This was accomplished by investigating two different PC cohorts of 10 units each. One of the cohorts, labelled as ‘Room Temperature’, was stored at 22℃±2℃ for 5 days and then transferred to a temperature of 4℃±2℃. The other cohort, labelled as ‘Cold’, was stored directly at 4℃±2℃. Both cohorts were stored for a total of 21 days and platelet indices, platelet counts, pH, and platelet factor IV (PF4) were measured at different time intervals. Sterility was performed on Day 21. The key findings showed no significant difference in mean platelet count, platelet distribution width (PDW), mean platelet volume (MPV), platelet-large cell ratio (P-LCR), and plateletcrit (PCT) between the two cohorts. On the other hand, a significant difference in mean pH and PF4 resulted between the two cohorts. Moreover, no significant difference in mean platelet count, and PCT was found between Days 1, 5, or 10, and Day 21 in both cohorts. However, an overall significant difference in mean PDW, MPV, P-LCR and pH was discovered between Days 1 and 21, Days 5 and 21, and Days 10 and 21 in both cohorts. Regards PF4, a significant difference was detected between Days 1 and 21, and Days 10 and 21; however, no significant difference was found between Days 5 and 21 in both cohorts. Corynebacterium freneyi and Microbacterium liquefaciens, were cultured from 1 unit of the room temperature cohort after it was flagged positive during the sterility testing. In conclusion, through the implementation of a delayed cold storage system, PCs can be safely administered to the patient.

Biosynthesis, Characterization and Antibacterial Properties of CuO Nanoparticles of Psychrotrophic Bacteria

Copper oxide nanoparticles have many applications in industry and medicine. Nevertheless, few studies have been conducted in relation to the biological production of these nanoparticles. In the present study, the ability of isolated Psychrotrophic (cold-resistant) prokaryotes from the Zagros highlands located in Lorestan province of Iran and some isolated Streptomyces strains from the Oman Sea has been evaluated in the synthesis of copper oxide nanoparticles. The maximum lethal concentration of copper salts was determined for the strains and the strains with the highest level of resistance were selected. The biosynthesis of copper oxide nanoparticles was done extracellularly by inoculating Psychrotrophic and Streptomyces strains in liquid TSB culture medium containing 0.01 M copper sulfate salt and keeping the culture medium in a shaker incubator at 20 °C at 150 rpm. The production of copper oxide nanoparticles was evaluated by changing the color of the reaction solution from light blue to dark green. From the 44 cold-resistant strains and the investigated Streptomyces strains, all strains were able to tolerate concentrations higher than 5 mM. Of these, the strain OSNP13 belonging to the genus Microbacterium liquefaciens (X77444) has been able to synthesize the highest amount of copper oxide nanoparticles. The optical absorption of the solution containing synthesized nanoparticles was determined in the range of 250-350 nm by UV-vis spectrophotometer, which had a specific peak at 288 nm. And the copper oxide nanoparticles have an average size of 63.21 nm based on DLS analysis. The crystallographic characteristics of copper oxide nanoparticles were also determined by using XRD analysis, which showed that the prepared nanoparticles had a hexagonal crystal structure with a size of 31.29 nanometers. Then, the antibacterial activity of produced nanoparticles was evaluated. The MIC values ​​of copper oxide nanoparticles for E. coli and S. aureus bacteria were calculated as 125 and 250 micrograms per milliliter, respectively. The produced copper oxide nanoparticles have shown a good antimicrobial properties and can be suitable candidates for use as antimicrobial agents.

CzcD gene from Bacillus megaterium and Microbacterium liquefaciens as a potential nickel-vanadium soil pollution biomarker.

Metals are among the most prevalent pollutants released into the environment. For these reasons, the use of biomarkers for environmental monitoring of individuals and populations exposed to metal pollution has gained considerable attention, offering fast and sensitive detection of chemical stress in organisms. There are different metal resistance genes in bacteria that can be used as biomarkers, including cation diffusion facilitators carrying metal ions; the prototype is the cobalt-zinc-cadmium transporter (czcD). The present study reports the expression changes in the czcD gene in Bacillus megaterium and Microbacterium liquefaciens under nickel and vanadium exposure by real-time polymerase chain reaction. The nickel-vanadium-resistant strains of B. megaterium and M. liquefaciens used in this study were isolated from mine tailings in Guanajuato, Mexico. The czcD gene showed high expression under exposure to 200 ppm of Ni and 200 ppm of V during the logarithmic growth phase of M. liquefaciens in PHGII liquid media. In contrast, no changes were observed in B. megaterium during logarithmic and stationary growth, perhaps due to the gene having differential expression during the growth phases. The expression profiles obtained for czcD show the possibility of using this gene from M. liquefaciens as a biomarker of nickel and vanadium pollution in microorganisms.

Biofilms and Extracts from Bacteria Producing "<i>Quorum Sensing</i>" Signaling Molecules Promote Chemotaxis and Settlement Behaviors in <i>Hydractinia symbiolongicarpus</i> (Cnidaria: Hydrozoa) Larvae

Many sessile marine invertebrates have life cycles involving the development of larvae that settle on specific substrates to initiate metamorphosis to juvenile forms. Although is recognized that bacterial biofilms play a role in this process, the responsible chemical cues are beginning to be investigated. Here, we tested the role of substrate-specific bacteria biofilms and their Quorum Sensing Signaling Molecule (QSSM) extracts on chemotaxis and settlement of larvae from Hydractinia symbiolongicarpus, a hydroid that grows on gastropod shells occupied by hermit crabs. We isolated and taxonomically identified by 16S rDNA sequencing, 14 bacterial strains from shells having H. symbiolongicarpus. Three isolates, Shigella flexneri, Microbacterium liquefaciens, and Kocuria erythromyxa, were identified to produce QSSMs using biosensors detecting N-acyl-L-homoserine lactones. Multispecies biofilms and QSSM extracts from these bacteria showed a positive chemotactic effect on H. symbiolongicarpus larvae, a phenomenon not observed with mutant strains of E. coli and Chromobacterium violaceum that are unable to produce QSSMs. These biofilms and QSSMs extracts induced high rates of larval attachment, although only 1 % of the attached larvae metamorphosed to primary polyps, in contrast to 99 % of larvae incubated with CsCl, an artificial inductor of attachment and metamorphosis. These observations suggest that bacterial QSSMs participate in H. symbiolongicarpus substrate selection by inducing larval chemotaxis and attachment. Furthermore, they support the notion that settlement in cnidarians is decoupled into two processes, attachment to the substrate and metamorphosis to a primary polyp, where QSSMs likely participate in the former but not in the latter.

Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry.

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion–substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein’s conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.

Expression Changes in Metal-Resistance Genes in Microbacterium liquefaciens Under Nickel and Vanadium Exposure.

Microbacterium liquefaciens MNSH2-PHGII-2 is a nickel-vanadium-resistant bacterium isolated from mine tailings located in Guanajuato, Mexico. In PHGII liquid media, M. liquefaciens has the ability to remove 29.5ppm of Ni and 168.3ppm of V. The present study reports, for the first time in M. liquefaciens, the presence of the genes nccA (Ni-Co-Cd resistance), hant (high-affinity nickel transporter), smtA, a metal-binding protein gene, and VAN2 (V resistance), which showed an increased expression under exposure to 200ppm of Ni and 200ppm of V during the logarithmic growth phase of the microorganism in PHGII liquid media. Data about the expression profile of genes conferring metal resistance to M. liquefaciens can improve the knowledge of those mechanisms involved in the processes of Ni-V resistance and probably in Ni-V removal processes. Based on our data, we can suggest that M. liquefaciens has the potential to be used in the biological treatment of toxic wastes with high Ni and V content.

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity.

This work reports the evolutionary relationships, amplified expression, functional characterization and purification of the putative allantoin transport protein, PucI, from Bacillus subtilis. Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between PucI and membrane proteins of the nucleobase-cation-symport-1 family of secondary active transporters. These include the sodium-coupled hydantoin transport protein, Mhp1, from Microbacterium liquefaciens, and related proteins from bacteria, fungi and plants. Membrane topology predictions for PucI were consistent with 12 putative transmembrane-spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. The pucI gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions determined for optimal amplified expression of the PucI(His6) protein in Escherichia coli to a level of about 5 % in inner membranes. Initial rates of inducible PucI-mediated uptake of 14C-allantoin into energized E. coli whole cells conformed to Michaelis-Menten kinetics with an apparent affinity (Kmapp) of 24 ± 3 μM, therefore confirming that PucI is a medium-affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that PucI recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His6) was solubilized from inner membranes using n-dodecyl-β-d-maltoside and purified. The isolated protein contained a substantial proportion of α-helix secondary structure, consistent with the predictions, and a 3D model was therefore constructed on a template of the Mhp1 structure, which aided localization of the potential ligand binding site in PucI.

Identification of Bacillus megaterium and Microbacterium liquefaciens genes involved in metal resistance and metal removal.

Bacillus megaterium MNSH1-9K-1 and Microbacterium liquefaciens MNSH2-PHGII-2, 2 nickel- and vanadium-resistant bacteria from mine tailings located in Guanajuato, Mexico, are shown to have the ability to remove 33.1% and 17.8% of Ni, respectively, and 50.8% and 14.0% of V, respectively, from spent petrochemical catalysts containing 428 ± 30 mg·kg(-1) Ni and 2165 ± 77 mg·kg(-1) V. In these strains, several Ni resistance determinants were detected by conventional PCR. The nccA (nickel-cobalt-cadmium resistance) was found for the first time in B. megaterium. In M. liquefaciens, the above gene as well as the czcD gene (cobalt-zinc-cadmium resistance) and a high-affinity nickel transporter were detected for the first time. This study characterizes the resistance of M. liquefaciens and B. megaterium to Ni through the expression of genes conferring metal resistance.

Microbacterium oxydans and Microbacterium liquefaciens: A biological alternative for the treatment of Ni-V-containing wastes

The present study evaluated 15 isolates obtained of environmental samples capable of tolerating high Ni and V concentrations. Those coded as MNSH2-PHGII-1, MNSH2-PHGII-2 and MV-PHGII-2 showed a minimum inhibitory concentration higher than 200 ppm for Ni and V and showed removal percentages corresponding to 84, 75 and 26% for Ni and 60, 55 and 20.3% for V, respectively, in liquid medium. When spent catalyst was added at 16% (w/v) pulp density, the highest Ni and V removal corresponded to MNSH2-PHGII-1 and MNSH2-PHGII-2, which were identified as Microbacterium oxydans and Microbacterium liquefaciens respectively, Microbacterium oxydans was able to remove Ni at the extent of 45.4% and V at 30.4% while Microbacterium liquefaciens removed Ni at 51% and V at 41.4% from the spent catalyst. The isolate MV-PHGII-2 identified also as Microbacterium oxydans showed the lowest removal for Ni and V corresponding to 16% and 9.5%, respectively. This is the first report where strains of Microbacterium were tested for their abilities to remove Ni and V from spent catalyst, suggesting its potential use in the treatment of this solid industrial waste.

Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

Modeling, Substrate Docking, and Mutational Analysis Identify Residues Essential for the Function and Specificity of a Eukaryotic Purine-Cytosine NCS1 Transporter

The recent elucidation of crystal structures of a bacterial member of the NCS1 family, the Mhp1 benzyl-hydantoin permease from Microbacterium liquefaciens, allowed us to construct and validate a three-dimensional model of the Aspergillus nidulans purine-cytosine/H(+) FcyB symporter. The model consists of 12 transmembrane α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct core of 10 TMSs is made of two intertwined inverted repeats (TMS1-5 and TMS6-10) that are followed by two additional TMSs. TMS1, TMS3, TMS6, and TMS8 form an open cavity that is predicted to host the substrate binding site. Based on primary sequence alignment, three-dimensional topology, and substrate docking, we identified five residues as potentially essential for substrate binding in FcyB; Ser-85 (TMS1), Trp-159, Asn-163 (TMS3), Trp-259 (TMS6), and Asn-354 (TMS8). To validate the role of these and other putatively critical residues, we performed a systematic functional analysis of relevant mutants. We show that the proposed substrate binding residues, plus Asn-350, Asn-351, and Pro-353 are irreplaceable for FcyB function. Among these residues, Ser-85, Asn-163, Asn-350, Asn-351, and Asn-354 are critical for determining the substrate binding affinity and/or the specificity of FcyB. Our results suggest that Ser-85, Asn-163, and Asn-354 directly interact with substrates, Trp-159 and Trp-259 stabilize binding through π-π stacking interactions, and Pro-353 affects the local architecture of substrate binding site, whereas Asn-350 and Asn-351 probably affect substrate binding indirectly. Our work is the first systematic approach to address structure-function-specificity relationships in a eukaryotic member of NCS1 family by combining genetic and computational approaches.

The alternating access mechanism of transport as observed in the sodium-hydantoin transporter Mhp1

Secondary active transporters move molecules across cell membranes by coupling this process to the energetically favourable downhill movement of ions or protons along an electrochemical gradient. They function by the alternating access model of transport in which, through conformational changes, the substrate binding site alternately faces either side of the membrane. Owing to the difficulties in obtaining the crystal structure of a single transporter in different conformational states, relatively little structural information is known to explain how this process occurs. Here, the structure of the sodium-benzylhydantoin transporter, Mhp1, from Microbacterium liquefaciens, has been determined in three conformational states; from this a mechanism is proposed for switching from the outward-facing open conformation through an occluded structure to the inward-facing open state.

Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1.

The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens comprises a five-helix inverted repeat, which is widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resolution, complementing its previously described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.

Molecular Basis of the “Alternate Access Model” in the Cation-Substrate Symporter Mhp1 from Computer Simulations and X-Ray Crystallography

A common structural motif is emerging for a wide class of substrate-cation symporters. The fold of the amino acid-sodium symporter LeuT is shared by proteins unrelated by sequence identity such as the galactose-sodium symporter vSGLT, the nucleobase-cation symporter Mhp1, the betaine-transporting osmoregulator BetP, and amino acid-proton transporters AdiC, and ApcT. The “alternating access” model explains transport as cycling between at least three distinct conformational states that connect a central binding site to either the extracellular or the intracellular compartment. The crystal structures solved so far can be broadly categorized in these three conformations, outward facing (LeuT, Mhp1, BetP, AdiC), occluded (Mhp1, BetP, ApcT), and inward facing (vSGLT). We are currently studying the crystal structure of Mhp1 hydantoin transporter from Microbacterium liquefaciens in the inward facing open state. Together with the previous structures [1] a full picture of the conformational change occurring during transport emerges. Dynamic importance (DIMS) molecular dynamics (MD) simulations allow us to connect these three states with continuous transition trajectories. The combination of structural and simulation data puts the alternate access model on a firm structural basis and will facilitate future detailed studies of the energetics of cation-substrate coupled transport.We acknowledge support from the BBSRC and the EU EDICT consortium.[1] Weyand et al. (2008) Science 322, 709-713.

Isolation of some pathogenic bacteria from the great spruce bark beetle, Dendroctonus micans and its specific predator, Rhizophagus grandis

Some bacteria were isolated from Dendroctonus micans and its specific predator, Rhizophagus grandis. Six bacteria from D. micans were identified as Bacillus pumilus, Enterobacter intermedius, Citrobacter freundii, Cellulomonas flavigena, Microbacterium liquefaciens and Enterobacter amnigenus, three bacteria from R. grandis as Klebsiella pneumoniae, Pantoea agglomerans and Serratia grimesii, on the basis of fatty acid methyl ester analysis and carbon utilization profile by using Microbial Identification and Biolog Microplate Systems. Their insecticidal effects were tested on larvae and adults of D. micans.

A Conserved Na+ Binding Site of the Sodium-coupled Neutral Amino Acid Transporter 2 (SNAT2)

The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na(+) into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT(Aa) and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na(+) binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na(+) binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na(+) binding to SNAT2, but also dramatically lowers the Na(+) affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na(+) binding and which is also expected to form part of the Na(+) binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na(+) affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.

Mutation of Asparagine 76 in the Center of Glutamine Transporter SNAT3 Modulates Substrate-induced Conductances and Na+ Binding

The glutamine transporter SLC38A3 (SNAT3) plays an important role in the release of glutamine from brain astrocytes and the uptake of glutamine into hepatocytes. It is related to the vesicular GABA (gamma-aminobutyric acid) transporter and the SLC36 family of proton-amino acid cotransporters. The transporter carries out electroneutral Na+-glutamine cotransport-H+ antiport. In addition, substrate-induced uncoupled cation currents are observed. Mutation of asparagine 76 to glutamine or histidine in predicted transmembrane helix 1 abolished all substrate-induced currents. Mutation of asparagine 76 to aspartate rendered the transporter Na+-independent and resulted in a gain of a large substrate-induced chloride conductance in the absence of Na+. Thus, a single residue is critical for coupled and uncoupled ion flows in the glutamine transporter SNAT3. Homology modeling of SNAT3 along the structure of the related benzyl-hydantoin permease from Microbacterium liquefaciens reveals that Asn-76 is likely to be located in the center of the membrane close to the translocation pore and forms part of the predicted Na+ -binding site.

Crystallization of the hydantoin transporter Mhp1 fromMicrobacterium liquefaciens

The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P2(1)2(1)2(1), unit-cell parameters a = 79.7, b = 101.1, c = 113.8 A). A complete data set has been collected from a single crystal to a resolution of 2.85 A with 64 741 independent observations (94% complete) and an R(merge) of 0.12. Further experimental phasing methods are under way.