Siderophore production of a periplasmic transport binding protein kinase gene defective mutant of Magnetospirillum magneticum AMB-1

A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis. The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed. The transposon was inserted in an open reading frame (ORF) coding for a periplasmic transport binding protein kinase gene homologue. Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon. Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotriacetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore. Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type. These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M. magneticum AMB-1.     

趋磁细菌AMB-1生物矿化相关蛋白Mms6参与磁小体的合成

【目的】研究趋磁细菌AMB-1生物矿化相关蛋白Mms6与磁小体合成的关系。【方法】在液体静置培养条件和好氧条件下对AMB-1进行培养,分析基因mms6在不同培养条件下转录水平的变化;对基因mms6进行基因敲除,分析突变株的生长和产磁变化。【结果】基因mms6的转录水平随着磁小体的合成逐渐升高;mms6的突变导致菌株在液体静置培养条件下趋磁性降低约50%,但不会影响菌株的生长水平。【结论】基因mms6参与了趋磁细菌AMB-1胞内磁小体的合成。     

Ligand-Dependent Structural Changes in the V1 ATPase from Manduca sexta

The response of V(1) ATPase of the tobacco hornworm Manduca sexta to Mg(2+) and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl(2)-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V(1) complex was supplemented with CuCl(2) nucleotide-dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP.PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V(1) complex. The catalytic subunit A was reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP.PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V(1) ATPase from M. sexta and conformational alterations in this enzyme due to Mg(2+) and nucleotide binding are discussed on the basis of these and previous observations.     

Interaction between subunit C (Vma5p) of the yeast vacuolar ATPase and the stalk of the C-depleted V1 ATPase from Manduca sexta midgut

Projection maps of a V₁-Vma5p hybrid complex, composed of subunit C (Vma5p) of Saccharomyces cerevisiae V-ATPase and the C-depleted V₁ from Manduca sexta, were determined from single particle electron microscopy. V₁-Vma5p consists of a headpiece and an elongated wedgelike stalk with a 2.1×3.0 nm protuberance and a 9.5×7.5 globular domain, interpreted to include Vma5p. The interaction face of Vma5p in V₁ was explored by chemical modification experiments.     

Functional analysis of spfD gene involved in DNA phosphorothioation in Pseudomonas fluorescens Pf0-1

DNA phosphorothioation is widespread in many bacterial species. By homology analysis of the dnd gene cluster in Pseudomonas fluorescens Pf0-1, a spfBCDE gene cluster involved in DNA phosphorothioation was localized. Disruption of the spfD gene, a dndD homolog, caused the loss of the Dnd phenotype and demonstrated the involvement of spfD in DNA phosphorothioation in P. fluorescens Pf0-1. The ATPase activity of SpfD suggests that SpfD could hydrolyze ATP to provide the energy required in the DNA phosphorothioate modification process.     

Solution NMR structure of CHU_1110 from Cytophaga hutchinsonii,an AHSA1 protein potentially involved in metal ion stress response

We report the solution nuclear magnetic resonance (NMR) structure of CHU_1110 from Cytophaga hutchinsonii. CHU_1110 contains three α-helices and one antiparallel β-sheet, forming a large cavity in the center of the protein, which are consistent with the structural characteristics of AHSA1 protein family. This protein shows high structural similarities to the prokaryotic proteins RHE_CH02687 from Rhizobium etli and YndB from Bacillus subtilis, which can bind with flavinoids. Unlike these two homologs, CHU_1110 shows no obvious interaction with flavonoids in NMR titration experiments. In addition, no direct interaction has been observed between CHU_1110 and ATP, although many homologous sequences of CHU_1110 have been annotated as ATPase. Combining the analysis of structural similarity of CHU_1110 and genomic context of its encoding gene, we speculate that CHU_1110 may be involved in the stress response of bacteria to heavy metal ions, even though its specific biological functions that need to be further investigated.     

CO2浓度升高对毛竹器官矿质离子吸收、运输和分配的影响

为了给大气CO2浓度逐渐升高背景下的毛竹林适应性经营管理提供理论依据,运用开顶式气室(OTCs)模拟大气CO2浓度升高(500、700 μmol/mol)情景,以目前环境背景大气为对照,研究了Na+、Fe2+-Fe3+、Ca2+、Mg2+等矿质离子在毛竹器官中吸收、运输和分配的变化规律.结果显示,除CO2浓度700 μmoL/mol对Ca2+浓度在毛竹器官中大小排序会产生影响外,CO2浓度500、700 μmol/mol并未改变毛竹器官中Na+、Fe2+,Fe3+、Mg2+、Ca2+浓度的大小排序.CO2浓度升高对竹叶Fe2+-Fe3+和竹枝Fe2+-Fe3+、Mg2+浓度无明显影响,但对器官的其它矿质离子浓度会有不同程度的影响,竹叶Ca2+和Mg2+、竹枝Na+和Ca2+、竹秆Na+和Ca2+及Mg2+、竹根Na+和Mg2+浓度明显提高,竹叶Na+、竹秆Fe2+-Fe3+、竹根Fe2+-Fe3+和Ca2+浓度明显降低;随着CO2浓度的升高,竹叶Fe2+-Fe3+/Na+、Mg2+/Na+和Ca2+/Na+,竹枝Ca2+/Mg2+及各器官Mg2+/Fe2+-Fe3+、Ca2+/Fe2+-Fe3+均逐渐增大,而竹枝、竹秆、竹根Fe2+-Fe3+/Na+、Mg2+/Na+、Ca2+/Na+和竹叶、竹秆、竹根Ca2+/Mg2+均逐渐减小;CO2浓度升高后除竹根-竹秆Sca.Na、竹秆-竹枝SMg,Fe和竹枝-竹叶Sca,Mg明显下降外,其余的毛竹器官矿质离子向上运输系数变化平缓或明显提高.研究表明CO2浓度升高增强了毛竹立竹根部积累Na+能力和Fe2+-Fe3+、Ca2+和Mg2+的向上选择性运输能力,提高了光合器官竹叶中矿质养分元素浓度,可维持体内矿质养分元素平衡,有利于提高毛竹对高浓度CO2环境的适应能力.     

Inositol phosphosphingolipid phospholipase C1 regulates plasma membrane ATPase (Pma1) stability in Cryptococcus neoformans

Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.     

The plant P1B-type ATPase AtHMA4 transports Zn and Cd and plays a role in detoxification of transition metals supplied at elevated levels

The transition metal Zn is essential for many physiological processes in plants, yet at elevated concentrations this, and the related non-essential metal Cd, can be toxic. Arabidopsis thaliana HMA4, belonging to the Type P1B subfamily of P-type ATPases, has recently been implicated in Zn nutrition, having a role in root to shoot Zn translocation. Using Arabidopsis insertional mutants, it is shown here that disruption of AtHMA4 function also results in increased sensitivity to elevated levels of Cd and Zn, suggesting that AtHMA4 serves an important role in metal detoxification at higher metal concentrations. AtHMA4 and a truncated form lacking the last 457 amino acids both confer Cd and Zn resistance to yeast but a mutant version of the full-length AtHMA4 (AtHMA4-C357G) does not; this demonstrates that the C-terminal region is not essential for this function. Evidence is presented that AtHMA4 functions as an efflux pump.     

The NMR solution structure of subunit G (G61-101) of the eukaryotic V1VO ATPase from Saccharomyces cerevisiae

Subunit G is an essential stalk subunit of the eukaryotic proton pump V₁V_O ATPase. Previously the structure of the N-terminal region, G_1-59, of the 13 kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G_61-101) of subunit G of the Saccharomyces cerevisiae V₁V_O ATPase. The protein forms an extended α-helix between residues 64 and 100, whereby the first five- and the last residues of G_61-101 are flexible. The surface charge distribution of G_61-101 reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G_1-59, the presented solution structure of G_61-101 enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V₁V_O ATPase.     

Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg^2 + dependent and catalyzes a similar S_N2" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give K_chorismate = 20 ± 4 μM, K_Mg^2 + = 294 ± 22 μM, K_L-gln = 11 ± 1 mM, and k_cat = 2.2 ± 0.2 s^− 1 for a random kinetic mechanism. PhzE can use NH₄⁺ as an alternative nucleophile, while Co^2 + and Mn^2 + are alternative divalent metals.     

Cycloastragenol,a triterpene aglycone derived from Radix astragali, suppresses the accumulation of cytoplasmic lipid droplet in 3T3-L1 adipocytes

Cycloastragenol (CAG), a bioactive triterpenoid sapogenin isolated from the Chinese herbal medicine Radix astragali, was reported to promote the phosphorylation of extracellular signal-regulated protein kinase (ERK). Here we investigated the effect of CAG on adipogenesis. The image-based Nile red staining analyses revealed that CAG dose dependently reduced cytoplasmic lipid droplet in 3T3-L1 adipocytes with the IC₅₀ value of 13.0 μM. Meanwhile, cytotoxicity assay provided evidence that CAG was free of injury on HepG2 cells up to 60 μM. In addition, using calcium mobilization assay, we observed that CAG stimulated calcium influx in 3T3-L1 preadipocytes with a dose dependent trend, the EC₅₀ value was determined as 21.9 μM. There were proofs that elevated intracellular calcium played a vital role in suppressing adipocyte differentiation. The current findings demonstrated that CAG was a potential therapeutic candidate for alleviating obesity and hyperlipidemia.