Only one gene is required for the glpT-dependent transport of sn-glycerol-3-phosphate in Escherichia coli

Summary Deletion and point mutants defective in the glpT-dependent sn-glycerol-3-phosphate transport system were isolated and located on the Escherichia coli chromosome. They mapped in glpT in the clockwise order gyrA, glpA, glpT at around 48 min on the Escherichia coli linkage map. The mutations within glpT were ordered by deletion mapping, three factor crosses, and by crosses involving transducing bacteriophages carrying glpT-lac operon fusions. Results obtained using these fusion phages indicated that glpT is transcribed in the counterclockwise direction on the E. coli linkage map.Complementation analysis using these mutants revealed only one complementation group. Thus, one gene is necessary and sufficient for the proton motive force-dependent sn-glycerol-3-phosphate transport system.     

Buchnera aphidicola (Aphid-Endosymbiont) glyceraldehyde-3-phosphate dehydrogenase: Molecular cloning and sequence analysis

Buchnera aphidicola is an endosymbiont of the aphid Schizaphis graminum. A 3.9-kb B. aphidicola DNA fragment was sequenced and found to contain two open reading frames (ORFs). The deduced amino acid sequence of one of the ORFs had an 85% identity to Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (Gap). Both of these proteins have a higher similarity to eukaryotic than to prokaryotic Gaps. The second ORF could not be readily identified. The sequence of the putative product indicated that it was a member of the family of ATP-binding, membrane-associated proteins. The highest amino acid identity (36%) was with E. coli FtsE, a protein involved in cell division.     

Molecular cloning and sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase gene from the violet root rot fungus, <Emphasis Type="Italic">Helicobasidium mompa</Emphasis>

The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was cloned from the violet root rot fungus, Helicobasidium mompa, and characterized. Only one copy of the gene was present in the genome of H. mompa. The H. mompa gpd gene was found to encode a protein of 335 amino acids and its putative protein showed high homology with GPD protein from other heterobasidiomycetes. Although the promoter region contains a TATA box and several common motifs, these sequence positions were very different from that of other basidiomycetes. Moreover, mRNA expression showed that the gpd gene was transcribed constitutively in H. mompa.     

Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.     

Compartmentation in the induction of the hexose-6-phosphate transport system of Escherichia coli

The induction of the hexose-6-phosphate transport system was investigated. Glucose-6-phosphate (G6P) at concentrations as low as 10(-4)m was able to induce this system in wild-type cells, as well as in mutants lacking phosphoglucose isomerase or G6P dehydrogenase. Growth in the presence of fructose-6-phosphate (F6P) induced the system only if the cells contained phosphoglucose isomerase. Furthermore, glucose and F6P were found to induce the system only if the extracellular concentration of G6P became appreciable in the medium as a consequence of the leakage of intracellular G6P formed from the glucose or F6P. Intracellular G6P was not an inducer even at high concentrations. The metabolism of glucose inhibited the induction of the hexose-6-phosphate transport system. Hypotheses for this compartmentalization of inducer and membrane-associated induction are presented.     

Purification, characterization, and cDNA sequence of glucose-6-phosphate dehydrogenase from potato (Solatium tuberosum L.)

Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) has been purified from potato tuber at least 850-fold to apparent homogeneity as judged by SDS-PAGE. The enzyme was characterized by K_m values of 260 μM for glucose-6-phosphate and 6 μM for NADP and a broad pH optimum between phi 7.5 and 9. NADPH, GTP, ATP, acetyl CoA and CoA inhibited G6PDH activity. Dithiothreitol (DTT) did not inactivate the enzyme. A highly specific antiserum was produced in a rabbit and used for immunodetection of G6PDH in Western blots. A cDNA library from potato leaves was screened with DNA probes produced by the polymerase chain reaction (PCR) in the presence of g6pdh-specific primers. A full-length cDNA clone was analyzed and the derived amino acid sequence compared with known G6PDH sequences from various sources. The homology of the plant sequence with G6PDH sequences from animals and yeast was found to be rather high (52%), whereas there was significantly lower homology with sequences of bacterial origin (37%). The lack of a plastidic signal sequence as well as the insensitivity of the recombinant enzyme towards reduced DTT, support the view that the cDNA sequence of a redox-independent cytosolic isoform was obtained.     

冬凌草1-脱氧-D-木酮糖-5-磷酸合成酶基因克隆与表达分析

1-脱氧-D-木酮糖-5-磷酸合成酶(1-deoxy-D-xylulose 5-phosphate synthase,DXS)是植物萜类代谢通路中2-C-甲基-D-赤藓糖醇-4-磷酸(MEP)途径的第一个关键酶,在植物萜类物质的生物合成中发挥重要的作用.为了研究该基因在冬凌草二萜类成分合成中的作用,该研究在冬凌草转录组测序结果的基础上设计一对特异性引物,采用RT-PCR方法得到冬凌草IrDXS基因cDNA全长序列,并对其蛋白进行理化性质分析、信号肽预测、亚细胞定位预测、蛋白质二级结构、三级结构预测分析及跨膜域分析等生物信息学分析,同时利用实时荧光定量PCR的方法检测IrDXS基因在冬凌草不同部位中的表达情况.结果表明:从冬凌草叶片中分离得到了一条编码DXS的全长基因,通过生物信息学软件分析发现,该基因编码全长2169 bp,编码722个氨基酸,分子量为77.7 kD.多序列比对发现该基因编码的蛋白和其他植物中已知的DXS蛋白序列具有较高的同源性,N端均包含了一段质体转运肽序列,并均具有一个保守的焦磷酸硫胺素结构域和与吡啶结合相关的DRAG结构域.序列进化树分析显示,IrDXS基因属于植物DXS2家族.DXS基因在冬凌草根中表达量最高、愈伤组织中最低.该研究首次获得了IrDXS基因的全长cDNA序列,并揭示了其在不同组织中的表达差异,为后续的深入研究IrDXS基因在冬凌草二萜类成分合成途径中的功能奠定了基础.     

Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (pIsC)

Summary An Escherichia coli strain deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity has previously been isolated, and the gene (plsC) has been shown to map near min 65 on the chromosome. I precisely mapped the location of plsC on the chromosome, and determined its DNA sequence. plsC is located between parC and sufI, and is separated from sufI by 74 bp. Upstream of plsC is parC, separated by 233 bp, which includes an active promoter. parC, plsC, and sufI are all transcribed in the counterclockwise direction on the chromosome, possibly in an operon with multiple promoters. The amino-terminal sequence of the partially purified protein, combined with the DNA sequence, reveal 1-acyl-sn-glycerol-3-phosphate acyltransferase to be a 27.5 kDa highly basic protein. The plsC gene product, 1-acyl-sn-glycerol-3-phosphate acyltransferase, is localized to the cytoplasmic membrane of the cell. The amino-terminal sequence of the purified protein reveals the first amino acid to be a blocked methionine residue, most probably a formyl-methionine. The amino acid sequence of 1-acyl-sn-glycerol-3-phosphate acyltransferase has a short region of homology to two other E. coli acyltransferases that utilize acyl-acyl carrier protein as the acyl donor, sn-glycerol-3-phosphate acyltransferase and UDP-N-acetyl-glucosamine acyltransferase (involved in lipid A biosynthesis).     

Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.     

The complete amino-acid sequence of the rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis

Summary SummarySeveral cDNA clones encoding the entire Rieske FeS-precursor protein of the chloroplast cytochrome b ₆ f-complex have been isolated by high density plaque immunoscreening of a phage lambda gt11 cDNA expression library, made from poly A⁺-RNA of spinach seedlings. The identity of the cDNAs has been confirmed by N-terminal amino acid sequencing of the purified protein. The nucleotide sequence indicates a protein of 247 amino acid residues including a putative transit sequence of 68 amino acids corresponding to molecular masses of 26.3 kDa (precursor) and 18.8 kDa (mature protein; 179 amino acid residues). Alignteins of the sequence with sequences from Rieske FeS-proteins of respiratory electron transport chains, two of bacterial and three of mitochondrial origin, shows little sequence homology, but remarkable similarity in secondary structure including a putative N-terminal transmembrane segment of about 25 residues and the peptides CTHLGCV and CPCHGS in the C-terminal region of the protein that are involved in the binding of the Fe₂S₂-cluster.     

Complete amino acid sequence of Bombyx egg-specific protein deduced from cDNA clone

Complementary (c)DNA coding for an insect yolk protein, the egg-specific protein of the silkworm Bombyx mori was cloned and the nucleotide sequence determined. The sequence covers the entire coding region of 1,677 base pairs with 5′ and 3′ noncoding regions (21 and 115 base pairs, respectively). The deduced amino acid sequence of the egg-specific protein consists of 559 amino acid residues. The NH₂-terminal 18 amino acid sequence is enriched in hydrophobic amino acids and assumed to be a signal peptide. A sequence, Asn-X-Thr, a potential N-linked glycosylation site, is found at positions 191 to 193. A serine-rich domain is localized in the region from 63 to 90, in which phosphorylation takes place. Cys His motif in 405 to 415 is analogous to a proposed metal binding sequence. Lys¹³²-Asn¹³³ and Arg²²⁸-Asp²²⁹ are probably the sites cleaved by the egg-specific protein protease that appears during embryogenesis. The derived amino acid sequence has no appreciable homology to other sequenced proteins.     

Cloning and nucleotide sequence of the chlD locus

The nucleotide sequence of a Sau3A1 restriction nuclease fragment that complemented an Escherichia coli chlD::Mu cts mutant strain was determined. DNA and deduced amino acid sequence analysis revealed two open reading frames (ORFs) that potentially codes for proteins with amino acid sequence homology with binding protein-dependent transport systems. One of the ORFs showed a sequence that encoded a protein with properties that were characteristic of a hydrophobic inner membrane protein. The other ORF, which was responsible for complementing a chlD mutant, encoded a protein with conserved sequences in nucleotide-binding proteins and hydrophilic inner membrane proteins in active transport systems. A proposal that the chlD locus is the molybdate transport operon is discussed in terms of the chlD phenotype.     

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene

Summary The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely –35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.     

The amino acid sequence of the alpha subunit of mouse salivary androgen-binding protein (ABP), with a comparison to the partial sequence of the beta subunit and to other ligand-binding proteins

It has been suggested that three distinct genes,Abpa, Abpb, andAbpg, determine the three subunits of mouse salivary androgen-binding protein (ABP) (Dlouhy, S. R.,et al., Genetics 115, 535, 1987). We report the putative amino acid sequence of the subunit common to all forms of ABP, the Alpha subunit, and the partial amino acid sequence of the Beta subunit. These sequences have little in common, supporting the notion of at least two distinct genes coding for the subunits of the most common form of salivary ABP, the A:B dimer. A search of GenBank showed that these sequences have not been reported previously. The Beta subunit shows significant homology with helospectin, a member of the glucagon superfamily, but not enough homology to assign it to the family. No homology exists between ABP subunits and members of the ligand-binding carrier family of proteins nor does ABP show homology with other androgen-binding proteins. Particularly interesting is the observation that there is no relationship to rat prostatic steroid binding protein (PBP), given the similarities in protein tertiary structure, the numbers of subunits and their genes, and the earlier observation of ABP cross-reactive material in mouse prostate.Partial support for this work was provided by a PHS AREA award and by the Butler Academic Grants program. Both sources of support are greatly appreciated.A portion of this work constituted partial fulfillment of the honors thesis requirement for Butler University.     

Identification and characterization of the exbB, exbD and tonB genes of Pseudomonas putida WCS358: their involvement in ferric-pseudobactin transport

Catechol-cephalosporins are siderophore-like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric-siderophore transport pathway. Mutants of strain WCS358 were isolated that are resistant to high concentrations of these antibiotics. These mutants failed to grow under iron-limiting conditions, and could not utilize different ferric-siderophores. The mutants fall in three complementation groups. The nucleotide sequence determination identified three contiguous open reading frames, which were homologous to the exbB, exbD and tonB genes of Escherichia coli respectively. The deduced amino acid sequence of P. putida ExbB showed 58.6% homology with its E. coli homologue, but, unlike the E. coli protein, it has a N-terminal extension of 91 amino acids. The ExbD proteins are 64.8% homologous, whereas the TonB proteins only show 27.7% homology. The P. putida exbB gene could complement an E. coli exbB mutation, but the TonB proteins were not interchangeable between the species. It is concluded that P. putida WCS358 contains an energy-coupling system between the membranes for active transport across the outer membrane, which is comprised of a TonB-like energy-transducing protein and two accessory proteins. This system is similar to, but not completely compatible with, the E. coli system.     

Cell envelope proteins involved in the transport of maltose and sn-glycerol-3-phosphate in Escherichia coli

Two types of proteins are discussed in their role of facilitating the transport of maltose and sn-glycerol-3-phosphate in E. coli. The first protein is the receptor for phage δ, known to be an outer membrane protein. By facilitating the diffusion of maltose and the higher maltodextrins through the outer membrane the effect of the δ receptor is to decrease the K_m of the transport system without influencing the V_max of substrate flux. The second protein is a periplasmic protein that is induced by growth on glycerol and is essential for transport of sn-glycerol-3-phosphate in whole cells but not in membrane vesicles. This protein has solely been identified by the use of a two-dimensional polyacrylamide gel electrophoresis of periplasmic proteins in wild-type and mutants defective in sn-glycerol-3-phosphate transport.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.