Cloning and expression of a full-length glutamate decarboxylase gene fromLactobacillus brevis BH2

A bacterium (BH2) that was found to produce a large amount of γ-aminobutyric acid (GABA) was isolated fromKimchi, a traditional fermented food in Korea. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that BH2 belonged to the genusLactobacillus brevis. Under controlled conditions in MRS broth (Difco) with 5% monosodium glutamate, this strain produced GABA at a concentration of 194 mM with a 73% GABA conversion rate after 48 h. A full-length glutamate decarboxylase (gad) gene was cloned by the rapid amplification of cDNA ends (RACE) PCR. The open reading frame (ORF) of thegad gene was composed of 1,407 nucleotides and encoded a protein (468 amino acids) with a predicted molecular weight of 53.5 kDa. The deduced amino acid sequence of GAD fromL. brevis showed 97.5 and 82.7% identities to theL. brevis OPK-3 GAD andL. plantarum WCFS1 GAD, respectively. Thegad gene was expressed inEscherichia coli cells and the expression was confirmed by SDS-PAGE analysis and enzyme activity studies.     

Chlorosome Proteins Studied by MALDI-TOF-MS: Topology of CsmA in Chlorobium tepidum

Chlorosomes, the light-harvesting apparatus of green bacteria, are a unique antenna system, in which pigments are organized in aggregates rather than associated with proteins. Isolated chlorosomes from the green sulphur bacterium Chlorobium tepidum contain 10 surface-exposed proteins. Treatment of chlorosomes from Chlorobium tepidum with protease caused changes in the spectral properties of bacteriochlorophyll c and digestion of chlorosome proteins. Using SDS-PAGE analysis, immunoblotting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) we have investigated the topology of the 59-residue CsmA protein. Our results show that at the N-terminus, the only amino acid available for protease degradation is the methionine. At the C-terminus, amino acids can be removed by protease treatment to produce a residual protein containing at least the sequence between residues 2 and 38. These results indicate that the N-terminal portion of the CsmA protein, which is predicted to be mainly hydrophobic, is buried in the chlorosome envelope.     

Pigment interactions in chlorosomes of various green bacteria

Resonance Raman experiments were performed on different green bacteria. With blue excitation, i.e. under Soret resonance or preresonance conditions, resonance Raman contributions were essentially arising from the chlorosome pigments. By comparing these spectra and those of isolated chlorosomes, it is possible to evaluate how the latter retain their native structure during the isolation procedures. The structure of bacteriochlorophyll oligomers in chlorosomes was interspecifically compared, in bacteriochlorophyllc- and bacteriochlorophylle- synthesising bacteria. It appears that interactions assumed by the 9-keto carbonyl group are identical inChlorobium limicola, Chlorobium tepidum, andChlorobium phaeobacteroides. In the latter strain, the 3-formyl carbonyl group of bacteriochlorophylle is kept free from intermolecular interactions. By contrast, resonance Raman spectra unambiguously indicate that the structure of bacteriochlorophyll oligomers is slightly different in chlorosomes fromChloroflexus auranticus, either isolated or in the whole bacteria.     

Isolation and characterization of the gene from Pseudomonas syringae pv. phaseolicola encoding the phaseolotoxin-insensitive ornithine carbamoyltransferase

Summary The gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) fromPseudomonas syringae pv.phaseolicola has been cloned and sequenced. The gene has a deduced coding capacity for a polypeptide with a calculated M, of 36520 daltons. Comparison of the amino acid sequence of the OCTase enzymes encoded by theP. aeruginosa argF and theEscherichia coli argI andargF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function. Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs. This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases. We propose that the gene be namedargK.     

Characterization of the csmD and csmE genes from Chlorobium tepidum. The CsmA, CsmC, CsmD, and CsmE proteins are components of the chlorosome envelope

The csmD and csmE genes, encoding two proteins of the chlorosome envelope, have been cloned and sequenced from the green sulfur bacterium Chlorobium tepidum. The csmD gene predicts a hydrophobic protein of 113 amino acids with a molecular mass of 11.1 kDa. The csmE gene was identified immediately upstream from csmD; the csmE gene predicts a protein of 82 amino acids (9.0 kDa) which is 49% identical to CsmA (Chung et al. (1994) Photosynthesis Res 41: 261–275). The CsmE protein is post-translationally processed, most likely in a manner similar to CsmA. The csmE and csmD genes are cotranscribed as a dicistronic mRNA but can also be cotranscribed with an open reading frame upstream from csmE that predicts a protein with sequence similarity to the CheY and SpoOF subclass of regulatory proteins. The CsmA, CsmC, CsmD, and CsmE proteins were overproduced in Escherichia coli, purified, and used to raise polyclonal antibodies in rabbits. Protease susceptibility mapping and agglutination experiments using these antibodies indicate that all four proteins are exposed at the surface of isolated chlorosomes and hence are probably components of the chlorosome envelope. Additionally, antigalactose antibodies were used to confirm that the galactosyl moiety of monogalactosyl diglycerol is exposed at the chlorosome surface; this is consistent with the notion that these lipids are components of the chlorosome envelope.Abbreviations BChl- bacteriochlorophyll - bp- basepair(s) - C.- Chloroflexus - Cb.- Chlorobium - Csm- chlorosome protein - csm gene encoding a chlorosome protein - IPTG isopropyl--d-thiogalatoside - nt nucleotide - ORF open reading frame - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae

Summary A DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA. Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa). A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance. Therefore, we termed it the ZRC1 (zinc resistance conferring) gene. The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions. While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn²⁺, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.     

Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas reinhardtii

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.Abbreviations OEE1, 2 and 3 oxygen evolution enhancer proteins 1, 2 and 3 - Rubisco ribulose bisphosphate carboxylase/oxygenase - PS photosystem - P21 and P30 C. reinhardtii thylakoid polypeptides 21 and 30     

Cloning and expression of antiviral/ribosome-inactivating protein from <Emphasis Type="Italic">Bougainvillea</Emphasis> x<Emphasis Type="Italic">buttiana</Emphasis>

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).     

Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine,serine and threonine

An intact cDNA fromArabidopsis thaliana for adenine phosphoribosyltransferase (APRT) was isolated and sequenced. The cDNA is 729 nucleotides in length and predicts a protein ofM _r 27140. The deduced amino acid sequence has been compared with those of other APRTs and shown to be most similar to theEscherichia coli protein. Construction of a molecular tree of the known APRT amino acid sequences indicates theA. thaliana andE. coli APRT sequences form one cluster and the currently available vertebrate and invertebrate sequences form a separate grouping. Since it is possible to select either for or against the expression of APRT, the isolation of this APRT cDNA clone will allow these selection schemes to be used in plant genetic experiments.     

Molecular cloning and functional expression of a novelNeurospora crassa xylose reductase inSaccharomyces cerevisiae in the development of a xylose fermenting strain

The development of a xylose-fermentingSaccharomyces cerevisiae yeast would be of great benefit to the bioethanol industry. The conversion of xylose to ethanol involves a cascade of enzymatic reactions and processes. Xylose (aldose) reductases catalyse the conversion of xylose to xylitol. The aim of this study was to clone, characterise and express a cDNA copy of a novel aldose reductase (NCAR-X) from the filamentous fungusNeurospora crassa inS. cerevisiae. NCAR-X harbours an open reading frame (ORF) of 900 nucleotides. This ORF encodes a protein (NCAR-X, assigned NCBI protein accession ID: XP_956921) consisting of 300 amino acids, with a predicted molecular weight of 34 kDa. TheNCAR-X-encoded aldose reductase showed significant homology to the xylose reductases ofCandida tenuis andPichia stipitis. WhenNCAR-X was expressed under the control of phosphoglycerate kinase I gene (PGK1) regulatory sequences inS. cerevisiae, its expression resulted in the production of biologically active xylose reductase. Small-scale oxygen-limited xylose fermentation with theNCAR-X containingS. cerevisiae strains resulted in the production of less xylitol and at least 15% more ethanol than the strains transformed with theP. stipitis xylose reductase gene (PsXYL1). TheNCAR-X-encoded enzyme produced byS. cerevisiae was NADPH-dependent and no activity was observed in the presence of NADH. The co-expression of theNCAR-X andPsXYL1 gene constructs inS. cerevisiae constituted an important part of an extensive research program aimed at the development of xylolytic yeast strains capable of producing ethanol from plant biomass.     

Vitellogenesis in both sexes of gonochoristic mud shrimp, Upogebia major (Crustacea): analyses of vitellogenin gene expression and vitellogenin processing

The mud shrimp, Upogebia major is a gonochoristic species with distinct sexual dimorphism; however, the male has the “ovarian part of testis” in the gonad and mature-looking eggs appear in a similar reproductive cycle to the female. Vitellogenesis of U. major was investigated focusing on the characterization of vitellogenin (Vg) gene expression and Vg processing. Vg cDNA cloned by PCR-based methods was 7799 bp-long, encoding 2568 amino acids in a single open reading frame. The deduced amino acid sequence shared common characteristics conserved in other shrimp Vgs. The Vg gene was expressed in the hepatopancreas of females and males, the ovary, and the ovarian part of testis. Vitellins (Vns) were detected in the gonads of both females and males as three prominent polypeptides with apparent molecular masses of 82 kDa, 100 kDa, and 115 kDa. N-terminal amino acid sequences determined for the three polypeptides were present in the deduced amino acid sequence, demonstrating that they derived from a single long Vg polypeptide. Immunoblot analysis using polyclonal antibodies raised against two Vns (82 kDa and 100 kDa) confirmed Vg processing in the hepatopancreas, in the hemolymph and possibly in the oocytes, similarly in both sexes.     

Characterization of csmB genes,encoding a 7.5-kDa protein of the chlorosome envelope,from the green sulfur bacteria Chlorobium vibrioforme 8327D and Chlorobium tepidum

The csmB gene, encoding the 7.5-kDa “Gerola-Olson” protein of chlorosomes, has been cloned and sequenced from the green sulfur bacteria Chlorobium vibrioforme strain 8327D and Chlorobium tepidum. Two potential start codons were identified, and the csmB gene may be translated into a preprotein with an amino-terminal extension. Two forms of the mature CsmB protein (74 or 75 amino acids in length) were identified that differ by the presence or absence of a methionine residue at the amino terminus. The csmB gene of Chl. tepidum is transcribed as an abundant monocistronic mRNA of approximately 350 nucleotides; primer extension mapping of the 5′ endpoint of the csmB mRNA suggests there is strong similarity between the csmB promoter and the σ⁷⁰ promoters of Escherichia coli. The CsmB protein of Chl. tepidum was overproduced as a histidine-tagged fusion protein in E. coli, purified to homogeneity by Ni²⁺ chelation affinity chromatography, and used to raise polyclonal antibodies in rabbits. Protease susceptibility mapping and agglutination experiments with isolated chlorosomes using anti-CsmB antibodies indicate that the CsmB protein is a component of the chlorosome envelope. Received: 28 May 1996 / Accepted: 17 July 1996     

Nucleotide Sequence of the Gene Encoding β-Isopropylmalate Dehydrogenase (leuB) from Bacteroides fragilis

The complete nucleotide sequence of the gene (leuB) coding for β-isopropylmaiate dehydrogenase of Bacteroides fragilis was determined. An open reading frame of 1,061 nucleotides was detected that could encode a polypeptide of 353 amino acid residues with a calculated molecular mass of 39,179 Da. The deduced amino acid sequence of the β-isopropylmalate dehydrogenase from B. fragilis showed substantial sequence similarity with the β-isopropylmalate dehydrogenases from other bacteria.     

Cloning and molecular characterization of the gene rimL which encodes an enzyme acetylating ribosomal protein L12 of Escherichia coli K12

Summary TherimL gene ofEscherichia coli K12 encodes en enzyme catalyzing the acetylation of the N-terminal serine of ribosomal protein L12, thereby converting it into L7. Using a mutant strain defective in this acetylation reaction, we cloned therimL gene into cosmid pHC79 and characterized it at the molecular level. From analysis by SDS-polyacrylamide gel electrophoresis of the proteins synthesized in maxi-cells containing derivatives of therimL-harboring plasmid into which transposon λδ had been inserted at various sites, the product of this gene was identified as a protein with an apparent molecular weight of 20.3 kDa. The nucleotide sequence of the gene and the amino acid sequence deduced from the nucleotide sequence were compared with those of two other ribosomal protein acetylses encoded by therimI andrimJ genes (Yoshikawa et al. 1987). A considerable degree of overall similarity was seen betweenrimL andrimJ, but the degree of similarity betweenrimL andrimI was very low. In addition, a short stretch of similar amino acid sequence was found in all threerim acetylases. The significance of these results with respect to other acetylating enzymes, in particular those involved in the acetylation of aminoglycoside antibiotics is discussed.