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PubMed1995

Existence of two ferredoxin-glutamate synthases in the cyanobacterium Synechocystis sp. PCC 6803. Isolation and insertional inactivation of gltB and gltS genes.

The first two genes of ferredoxin-dependent glutamate synthase (Fd-GOGAT) from a prokaryotic organism, the cyanobacterium Synechocystis sp. PCC 6803, were cloned in Escherichia coli. Partial sequencing of the cloned genomic DNA, of the 6.3 kb Hind III and 9.3 kb Cla I fragments, confirmed the existence of two different genes coding for glutamate synthases, named gltB and gltS. The gltB gene was completely sequenced and encodes for a polypeptide of 1550 amino acid residues (M(r) 168,964). Comparative analysis of the gltB deduced amino acid sequence against other glutamate synthases shows a higher identity with the alfalfa NADH-GOGAT (55.2%) than with the corresponding Fd-GOGAT from the higher plants maize and spinach (about 43%), the red alga Antithamnion sp. (42%) or with the NADPH-GOGAT of bacterial source, such as Escherichia coli (41%) and Azospirillum brasilense (45%). The detailed analysis of Synechocystis gltB deduced amino acid sequence shows strongly conserved regions that have been assigned to the 3Fe-4S cluster (CX5CHX3C), the FMN-binding domain and the glutamine-amide transferase domain. Insertional inactivation of gltB and gltS genes revealed that both genes code for ferredoxin-dependent glutamate synthases which were nonessential for Synechocystis growth, as shown by the ferredoxin-dependent glutamate synthase activity and western-blot analysis of the mutant strains.

Amino Acid Oxidoreductases↗

PubMed1992

Conserved structural and regulatory regions in the Salmonella typhimurium btuB gene for the outer membrane vitamin B12 transport protein.

The Salmonella typhimurium btuB gene encodes an outer membrane protein that is necessary for the transport of vitamin B12 and the uptake of the E colicins and bacteriophage BF23. The sequence of this gene showed 87% identity of the deduced polypeptide to its Escherichia coli homolog, and its product was found to function in transport as effectively in cells of E. coli as did the native protein. The extensive sequence conservation within the first 300 transcribed nucleotides, which include the leader and early part of the coding sequence, supports the proposed role of this region in the regulation of btuB gene expression.

Bacterial Outer Membrane Proteins↗

PubMed2001

Dynamic flexibility in the Escherichia coli genome.

Empirical rules based on tetranucleotide parameters were presented to predict the structural parameters twist (Omega), roll (rho), tilt (tau) and slide (D(y)). A statistical mechanical model was used to analyze the flexibility of the Escherichia coli genome. The replication terminus region displayed a low level of flexibility. A strong correlation can be seen between G+C content and flexibility. Average flexibilities in the coding regions were found to be significantly larger than those in non-coding regions. The flexible characteristics in the 5'-neighborhood of the coding regions and in three class sigma promoter sequences in the E. coli genome were also analyzed.

Escherichia coli↗

PubMed1982

Mitochondrial L-rRNA from Aspergillus nidulans: potential secondary structure and evolution.

The alignment of gene sequences coding for A. nidulans mitochondrial L-rRNA and E. coli 23S rRNA indicates a strong conservation of primary and potential secondary structure of both rRNA molecules, except that homologies to the 5'-terminal 5.8S-like region and the 3'-terminal 4.5S-like region of bacterial rRNA are not detectable on mtDNA. The structural organization of the A. nidulans mt L-rRNA gene corresponds to that of yeast omega + strains: both genes are interrupted by a large intron sequence (1678 and 1143 bp, respectively) and by another smaller insert (91 and 66 bp) at homologous positions within domain V. An evolutionary tree derived from conserved L-rRNA gene sequences of yeast nuclei, E. coli, maize chloroplasts and six mitochondrial species exhibits a common root of organelle and bacterial sequences separating early from the nuclear branch.

Animals↗

PubMed1997

Purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in Escherichia coli.

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function(s) remains unclear. A more complete analysis of the physiological activities of this oncofetal protein has, until now, been severely limited by the lack of an appropriate source from which to obtain pure AFP in any sizeable quantity. In the present investigation, we obviate this problem by cloning and efficiently overexpressing mature mouse and human AFP cDNA's in Escherichia coli. For recombinant mouse AFP (rMoAFP), large segments of the coding region were excised from the preexisting plasmids pAFP1 and pAFP2, which together encompass 90% of the AFP sequence. The mouse cDNA was made complete by the addition of N- and C-terminal encoding oligonucleotides. Mouse AFP cDNA was expressed directly as a full-length molecule in vector pTrp4 or as fusion proteins in plasmids pMALc and pRX1 under the transcriptional control of trp or tac promoters. Accumulation of rMoAFP was significantly increased in protease-deficient E. coli strains over nonprotease-deficient strains, > or = 10% of total cell protein. Of the gene fusion proteins examined, none offered significant advantage over the direct expression product in terms of recombinant protein stability, overall levels of synthesis, or facilitated purification. Recombinant AFP polypeptides expressed by pTrp4 were as expected, deposited in bacterial inclusion bodies. Subsequent to resolubilization/refolding, rMoAFP was first enriched by passage over Q-Sepharose resin followed by final purification using immobilized copper-chelate affinity chromatography. Protein sequencing of the N-terminus revealed that purified rMoAFP had a deletion of the first nine amino acids coded for by the full-length mouse AFP cDNA. Similar N-terminal deletions are observed with AFP isolates originating from natural sources. A complete human AFP cDNA was generated from a fetal liver cDNA library and was cloned into vector pTrp4. Recombinant human AFP (rHuAFP) was expressed under the identical conditions employed for rMoAFP but purification had to be modified to include preparative Mono Q anion exchange chromatography. N-terminal sequencing, amino acid compositional analysis, and electrospray mass spectrometry revealed that purified rHuAFP was intact and unaltered and that the initiator methionine was completely removed. The biological activity of recombinant AFP, as judged by its inhibitory effects on in vitro lymphocyte proliferation, was equivalent to that of the native protein. The availability of large quantities of mouse and human recombinant AFP molecules should now permit detailed structure-function analyses of this important oncofetal protein to proceed in a manner unimpeded by previous limitations in both quantity and quality of the native proteins.

Amino Acid Sequence↗

PubMed2003

The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana.

The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost identical to that of Arabidopsis: However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis: Non-coding regions are mostly divergent between the two plants. One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss.

Amino Acid Sequence↗

PubMed1990

Sequence and relatedness in other bacteria of the Pseudomonas aeruginosa oprP gene coding for the phosphate-specific porin P.

The oprP gene encoding the Pseudomonas aeruginosa phosphate-specific outer membrane porin protein OprP was sequenced. Comparison of the derived amino acid sequence with the known sequences of other bacterial porins demonstrated that OprP could be no better aligned to these porin sequences than it could to the periplasmic phosphate-binding protein PhoS of Escherichia coli. Southern hybridization and restriction mapping of the oprP gene in 37 clinical isolates and the 17 serotype strains of P. aeruginosa revealed that restriction sites in the vicinity of the oprP gene were highly conserved. Several species from the Pseudomonas fluorescens rRNA homology group contained DNA that hybridized to an oprP gene probe.

Amino Acid Sequence↗

PubMed1995

Developmental and pathogen-induced activation of an msr gene, str 246C, from tobacco involves multiple regulatory elements.

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5' flanking DNA sequence from the str246C gene fused to the beta-glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5' deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 bp was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.

Base Sequence↗

PubMed1997

Sequencing of a 1,3-1,4-beta-D-glucanase (lichenase) from the anaerobic fungus Orpinomyces strain PC-2: properties of the enzyme expressed in Escherichia coli and evidence that the gene has a bacterial origin.

A 971-bp cDNA, designated licA, was obtained from a library of Orpinomyces sp. strain PC-2 constructed in Escherichia coli. It had an open reading frame of 738 nucleotides encoding LicA (1,3-1,4-beta-D-glucanase; lichenase) (EC 3.2.1.73) of 245 amino acids with a calculated molecular mass of 27,929 Da. The deduced amino acid sequence had high homology with bacterial beta-glucanases, particularly in the central regions and toward the C-terminal halves of bacterial enzymes. LicA had no homology with plant beta-glucanases. The genomic DNA region coding for LicA was devoid of introns. More than 95% of the recombinant beta-glucanase produced in E. coli cells was found in the culture medium and periplasmic space. A N-terminal signal peptide of 29 amino residues was cleaved from the enzyme secreted from Orpinomyces, whereas 21 amino acid residues of the signal peptide were removed when the enzyme was produced by E. coli. The beta-glucanase produced by E. coli was purified from the culture medium. It had a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gels. The Km and Vmax values with lichenin as the substrate at pH 6.0 and 40 degrees C were 0.75 mg/ml and 3,790 micromol/min/mg, respectively. With barley beta-glucan as the substrate, the corresponding values were 0.91 mg/ml and 5,320 micromol/min/mg. This enzyme did not hydrolyze laminarin, carboxymethylcellulose, pustulan, or xylan. The main products of lichenin and barley beta-glucan hydrolysis were triose and tetraose. LicA represented the first 1,3-1,4-beta-D-glucanase reported from fungi. The results presented suggest that licA of Orpinomyces had a bacterial origin.

Amino Acid Sequence↗

PubMed1991

Sequence analysis of the ARG7 gene of Schizosaccharomyces pombe coding for argininosuccinate lyase. Expression of the gene in Saccharomyces cerevisiae.

The complete nucleotide sequence of the ARG7 gene, coding for argininosuccinate lyase (EC 4.3.2.1), in the fission yeast (Schizosaccharomyces pombe) has been determined. It consists of an open reading frame of 461 codons. The deduced protein has a molecular weight of 51,200 Da. The gene is devoid of introns which is confirmed by the fact that it is expressed in Escherichia coli after spontaneous insertion of a bacterial sequence probably bearing a prokaryotic promoter. A perfect "TATA" box is found at -72 and the major transcription initiation site in Saccharomyces cerevisiae is located at -11 as shown by primer extension experiments. Comparison of the S. pombe lyase with related proteins from other organisms reveals an important degree of conservation except in the carboxyterminal part of the polypeptide. Additionally, a deletion removing 66 amino acids of the carboxy terminus yields an enzyme exhibiting some biological activity. A unique 1,500 b transcript was found in S. cerevisiae when the intact gene was present, but the deleted version of the gene gave rise to at least three transcripts of 1,800, 2,800 and 3,900 b.

Amino Acid Sequence↗

PubMed1985

Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli.

The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage lambda D69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed by lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells and the subunit molecular mass established as 33,000 daltons. Analysis, by the minicell procedure, of plasmids with deletions extending into the prs gene established the direction of transcription as counterclockwise. A putative leader sequence of approximately 400 bp preceded the coding sequence. By deletion analysis and by cloning fragments of this leader sequence in a galK expression vector it was found to contain the prs promoter as well as a potential transcription termination site.

Cloning, Molecular↗

PubMed1991

Cloning an Escherichia coli gene encoding a protein remarkably similar to mammalian aldehyde dehydrogenases.

The nucleotide (nt) sequence of 2.9 kb of Escherichia coli genomic DNA that encompasses a gene encoding a putative aldehyde dehydrogenase (ALDH) has been determined. The presence of an open reading frame beginning 2 nt downstream from the ALDH-coding sequence indicates that this gene may be part of a larger operon. An extended upstream nt sequence displays striking similarity to sequences found upstream or in intergenic regions of a number of other bacterial genes. Crude cell extracts from E. coli grown under several different conditions show weak but measurable ALDH enzyme activity that prefers NADP+ over NAD+ as coenzyme; however, aldH gene expression appears to be very low, since no specific transcripts derived from the novel gene can be detected on Northern blots of RNA isolated from these cells. The deduced E. coli protein contains 495 amino acid (aa) residues with a calculated Mr of 53418. Its aa sequence showed marked similarity to NAD(+)-dependent ALDHs of eukaryotes. About 40% aa sequence identity, and over 60% similarity, are detected between the E. coli protein and both the cytosolic (class-1) and the mitochondrial (class-2) forms of mammalian ALDHs. In contrast to the mammalian enzymes, which contain eight to eleven Cys residues, only four Cys are present in the E. coli protein, and of these only Cys302, corresponding to the disulfiram-sensitive residue in the mammalian enzymes, is found at a conserved position in both the prokaryotic and the eukaryotic ALDHs. The availability of a bacterial ALDH with a high degree of similarity to mammalian ALDHs promises to facilitate future structural studies on these enzymes.

Aldehyde Dehydrogenase↗

PubMed2000

Mouse connexin37: gene structure and promoter analysis.

Connexin37 (Cx37) is a subunit gap junction protein which exhibits limited expression in only a few cell types, predominantly in endothelial cells and in the lung. To begin to analyze Cx37 expression, we isolated a 1.6 kb mouse Cx37 cDNA from a mouse lung cDNA library and isolated corresponding mouse genomic clones from a bacterial artificial chromosome library. Sequencing and comparison of these clones showed that the Cx37 gene contained a short first exon, an 1.0 kb single intron and a second exon containing the complete coding region and 3'-untranslated region (UTR). The 5'-UTR of the mouse cDNA showed 70% identity to that of human Cx37. Primer extension experiments performed using mouse lung RNA gave two bands of sizes consistent with the transcription start site predicted from the cDNA. Sequence analysis showed that the regions flanking exon I contained a consensus 'TATA box' 43 bp 5' from the transcription start site preceded by several putative transcription factor binding sites and a 282 bp truncated L1Md interspersed element. Luciferase reporter gene transfections suggested that an area of 268 bp 5' from the first exon acted as a basal promoter for Cx37 and that there was a strong negative regulatory element in the intron.

3' Untranslated Regions↗

PubMed2002

A real-time polymerase chain reaction method for monitoring anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene.

We have developed a real-time polymerase chain reaction (PCR) method that can quantify hydrocarbon-degrading bacteria in sediment samples based on a catabolic gene associated with the first step of anaerobic toluene and xylene degradation. The target gene, bssA, codes for the alpha-subunit of benzylsuccinate synthase. The primer-probe set for real-time PCR was based on consensus regions of bssA from four denitrifying bacterial strains; bssA sequences for two of these strains were determined during this study. The method proved to be sensitive (detection limit ca. 5 gene copies) and had a linear range of >7 orders of magnitude. We used the method to investigate how gasohol releases from leaking underground storage tanks could affect indigenous toluene-degrading bacteria. Microcosms inoculated with aquifer sediments from four different sites were incubated anaerobically with BTEX (benzene, toluene, ethylbenzene, and xylenes) and nitrate in the presence and absence of ethanol. Overall, population trends were consistent with observed toluene degradation activity: the microcosms with the most rapid toluene degradation also had the largest numbers of bssA copies. In the microcosms with the most rapid toluene degradation, numbers of bssA copies increased 100-to 1000-fold over the first 4 days of incubation, during which time most of the toluene had been consumed. These results were supported by slot blot analyses with unamplified DNA and by cloning and sequencing of putative bssA amplicons, which confirmed the real-time PCR method's specificity for bssA. Use of a companion real-time PCR method for estimating total eubacterial populations (based on 16S rDNA) indicated that, in some cases, ethanol disproportionately supported the growth of bacteria that did not contain bssA. The real-time PCR method for bssA could be a powerful tool for monitored natural attenuation of BTEX in fuel-contaminated groundwater. To our knowledge, this is the first reported molecular method that targets anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene.

Biodegradation, Environmental↗

PubMed2003

Antibacterial peptides: basic facts and emerging concepts.

Antibacterial peptides are the effector molecules of innate immunity. Generally they contain 15-45 amino acid residues and the net charge is positive. The cecropin type of linear peptides without cysteine were found first in insects, whilst the defensin type with three disulphide bridges were found in rabbit granulocytes. Now a database stores more than 800 sequences of antibacterial peptides and proteins from the animal and plant kingdoms. Generally, each species has 15-40 peptides made from genes, which code for only one precursor. The dominating targets are bacterial membranes and the killing reaction must be faster than the growth rate of the bacteria. Some antibacterial peptides are clearly multifunctional and an attempt to predict this property from the hydrophobicity of all amino acid side chains are given. Gene structures and biosynthesis are known both in the fruit fly Drosophila and several mammals. Humans need two classes of defensins and the cathelicidin-derived linear peptide LL-37. Clinical cases show that deficiencies in these peptides give severe symptoms. Examples given are morbus Kostmann and atopic allergy. Several antibacterial peptides are being developed as drugs.

Animals↗

PubMed2000

A novel 3-methyladenine DNA glycosylase from Helicobacter pylori defines a new class within the endonuclease III family of base excision repair glycosylases.

The cloning, purification, and characterization of MagIII, a 3-methyladenine DNA glycosylase from Helicobacter pylori, is presented in this paper. Sequence analysis of the genome of this pathogen failed to identify open reading frames potentially coding for proteins with a 3-methyladenine DNA glycosylase activity. The putative product of the HP602 open reading frame, reported as an endonuclease III, shares extensive amino acid sequence homology with some bacterial members of this family and has the canonic active site helix-hairpin-helix-GPD motif. Surprisingly, this predicted H. pylori endonuclease III encodes a 25,220-Da protein able to release 3-methyladenine, but not oxidized bases, from modified DNA. MagIII has no abasic site lyase activity and displays the substrate specificity of the 3-methyladenine-DNA glycosylase type I of Escherichia coli (Tag) because it is not able to recognize 7-methylguanine or hypoxanthine as substrates. The expression of the magIII open reading frame in null 3-methyladenine glycosylase E. coli (tag alkA) restores to this mutant partial resistance to alkylating agents. MagIII-deficient H. pylori cells show an alkylation-sensitive phenotype. H. pylori wild type cells exposed to alkylating agents present an adaptive response by inducing the expression of magIII. MagIII is thus a novel bacterial member of the endonuclease III family, which displays biochemical properties not described for any of the members of this group until now.

Amino Acid Motifs↗

PubMed1991

Structure of the beta-glucosidase gene bglA of Clostridium thermocellum. Sequence analysis reveals a superfamily of cellulases and beta-glycosidases including human lactase/phlorizin hydrolase.

The nucleotide sequence of the Clostridium thermocellum gene bglA, coding for the thermostable beta-glucosidase A, has been determined. The coding region of 1344 bp was identified by comparison with the N-terminal amino acid squence of recombinant beta-glucosidase A purified from Escherichia coli. The deduced amino acid sequence corresponds to a protein of 51,482 Da. The coding region is flanked by putative promoter and transcription terminator sequences. The protein is unrelated to beta-glucosidase B of C. thermocellum, but has a high level of similarity with other bacterial beta-glucosidases and phospho-beta-glucosidases. Similarity is also observed with the beta-galactosidase of the archaebacterium Sulfolobus solfataricus. Unexpectedly, it was found that human lactase-phlorizin hydrolase contains three copies of a sequence closely related to C. thermocellum beta-glucosidase A (up to 40% sequence identity). These diverse beta-glucosidases can therefore be grouped into an enzyme family (BGA) of common structural design. Sequence comparison by hydrophobic cluster analysis revealed that all BGA enzymes share a well conserved region which is homologous to the catalytic domain of the widely distributed cellulase family A. A distinctive feature of this domain is the sequence motif His-Asn-Glu-Pro in which the catalytic residues His and Glu are separated by 35-55 amino acid residues. The cellulase family A and the beta-glucosidase family BGA might thus be considered as members of a protein super-family comprising beta-glucanases and beta-glycosidases from all three primary kingdoms of living organisms.

Amino Acid Sequence↗

PubMed1995

Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains.

Peptide synthetases involved in the nonribosomal synthesis of peptide secondary metabolites possess a highly conserved domain structure. The arrangement of these domains within the multifunctional enzymes determines the number and order of the amino acid constituents of the peptide product. A general approach has been developed for targeted substitution of amino acid-activating domains within the srfA operon, which encodes the protein templates for the synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. Exchange of domain-coding regions of bacterial and fungal origin led to the construction of hybrid genes that encoded peptide synthetases with altered amino acid specificities and the production of peptides with modified amino acid sequences.

Amino Acid Sequence↗