Polyamines as modulators of gene expression under oxidative stress in Escherichia coli

Activity of enzymes of polyamine synthesis and contents of their products increased in E. coli cells in response to oxidative stress caused by addition of hydrogen peroxide to an exponentially growing culture. Putrescine and spermidine added to the culture medium in physiological concentrations significantly increased expression of genes oxyR and katG responsible for defense against oxidative stress, whereas cadaverine had no effect. The role of polyamines as modulators of the gene expression was confirmed by experiments with an inhibitor of polyamine synthesis, 1,3-diaminopropane, which decreased the level of cell polyamines and thus abolished the ability of the cell to induce oxyR expression under oxidative stress. A genetic method gave similar results: under oxidative stress mutants with disorders in polyamine synthesis displayed a significantly decreased level of induction of the oxyR and katG genes, and this level was recovered on addition of putrescine. In the presence of inhibitors of DNA-gyrase, nalidixic acid and novobiocin, the oxyR expression depended on the extent of DNA supercoiling. Putrescine decreased the inhibitory effects of nalidixic acid and novobiocin, and this confirmed its properties of a stimulator of DNA supercoiling. Resistance to rifampicin was studied to exemplify the mutation rate under oxidative stress. Putrescine decreased twofold the level of mutations and increased the number of viable cells in the culture exposed to oxidative stress.     

Isolation of oxidative stress response mutants in Escherichia coli for their use as a genetic screen to identify new anti-mycobacterials as functional analogues of isoniazid

Summary Tuberculosis is a leading killer disease of the world with increasing mortality due to HIV-infected individuals becoming highly prone to this infection. An attempt has been made in the present work to identify novel plant-derived compounds active against Mycobacterium tuberculosis (MTB) through construction of a target based bio-screen to facilitate rapid screening of anti-TB plant compounds. To achieve this, construction of a genetically modified model system was attempted in fast growing, non-pathogenic, Escherichia coli in which experimental testing is relatively easier and rapid as compared to M. tuberculosis, which is pathogenic and slow growing in nature. The exquisitely high sensitivity of M. tuberculosis to isoniazid (INH) has been attributed to lesions in oxyR, a gene that positively regulates the expression of a set of hydrogen peroxide-inducible genes in E. coli and S. typhimurium. Moreover in the mechanism of emergence of INH resistance in M. tuberculosis, oxidative stress response has been implicated. In this study, mutants of E. coli defective in oxidative stress response function were derived and used to screen plant compounds, which might interfere with the oxidative stress response in MTB. Since MTB is inherently known to be oxyR defective and thus being highly sensitive to INH, mutants defective in oxidative stress response were isolated to construct a model system in E. coli, which is otherwise INH resistant, having functional oxyR. These mutants showed simultaneous sensitivity to oxidative stress-causing agents like hydrogen peroxide and cumene hydroperoxide. To further define the mutational lesions, complementation studies were carried out through mobilization of cloned wild type genes involved in the oxidative stress response and in this way a biological screen was constructed to identify plant compounds/essential oils/extracts/oil components which induce oxidative stress. The positives were finally tested for activity against M. tuberculosis strain H37Rv using the radiometric BACTEC 460 TB system. Interestingly, the bioactives were found to be active against the pathogen with marked potency, as the reduction in δGI values for the identified bioactives against M. tuberculosis were significant. The study demonstrates application of a specific target-based genetic model system in E. coli as a rapid high throughput screen in identifying anti-mycobacterials from plants.     

The dcr gene family of Desulfovibrio: Implications from the sequence of dcrH and phylogenetic comparison with other mcp genes

Desulfovibrio vulgaris Hildenborough contains a family of genes for methyl-accepting chemotaxis proteins (MCPs). Here we report the complete sequence of the gene for Desulfovibrio chemoreceptor H (dcrH). The deduced amino acid sequence of DcrH protein, which has an enlarged N-terminal, ligand binding domain, indicates a structure similar to that of other MCPs. Comparison of the sequences for DcrA, determined earlier, and DcrH indicated that similarity is essentially limited to the C-terminal excitation region. The dcr gene family differs, in this respect, from mcp gene families in other eubacteria (e.g. Escherichia coli and Bacillus subtilis), where MCPs share significant homology throughout their C-terminal signal transduction domains. This may point to an ancient evolutionary origin of the dcr gene family, which is widely distributed throughout the genus Desulfovibrio. The evolutionary origin of mcp genes was traced by comparing nucleotide sequences for the excitation region that is common to all MCPs. Phylogenetic analysis of sequences for thirty mcp genes from nine eubacterial and one archaebacterial species suggested that multiplication of mcp genes has occurred at least twice since the eubacteria diverged from the archaebacteria.Nucleotide accession number: The nucleotide sequence reported in this paper has been entered into GenBank under accession number U30319. Phone: 403-220-6388. Fax: 403-289-9311. Electronic mail address: voordouw@acs.ucalgary.ca.     

Evolutionary relationships between catabolic pathways for aromatics: Conservation of gene order and nucleotide sequences of catechol oxidation genes of pWW0 and NAH7 plasmids

Summary TOL plasmid pWW0 and plasmid NAH7 encode catabolic enzymes required for oxidative degradation of toluene and naphthalene, respectively. The gene order of the catabolic operon of NAH7 for salicylate oxidation was determined to be: promoter-nahG (the structural gene for salicylate hydroxylase)-nahH (catechol 2,3-dioxygenase)-nahI (hydroxymuconic semialdehyde dehydrogenase)-nahN (hydroxymuconic semialdehyde hydrolase)-nahL (2-oxopent-4-enoate hydratase). This order is identical to that of the isofunctional genes of TOL plasmid pWW0. The complete nucleotide sequence of nahH was determined and compared with that of xylE, the isofunctional gene of TOL plasmid pWW0. There were 20% and 16% differences in their nucleotide and amino acid sequences, respectively. The homology between the NAH7 and TOL pWW0 plasmids ends upstream of the Shine-Dalgarno sequences of nahH and xylE, but the homology continues downstream of these genes. This observation suggested that genes for the catechol oxidative enzymes of NAH7 and TOL pWW0 were derived from a common ancestral sequence which was transferred as a discrete segment of DNA between plasmids.     

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.     

A PMR2 tandem repeat with a modified C-terminus is located downstream from the KRS1 gene encoding lysyl-tRNA synthetase in Saccharomyces cerevisiae

Summary TheKRS1 gene encodes the cytoplasmic form ofSaccharomyces cerevisiae lysyl-tRNA synthetase. TheKRS1 locus has been characterized. The lysyl-tRNA synthetase gene is unique in the yeast genome. The gene is located on the right arm of chromosome IV and disruption of the open reading frame leads to lethality. These results contrast with the situation encountered inEscherichia coli where lysyl-tRNA synthetase is coded by two distinct genes,lysS andlysU, and further address the possible biological significance of this gene duplication. The nucleotide sequence of the 3′-flanking region has been established. It encodes a long open reading frame whose nucleotide and amino acid structures are almost identical toPMR2, a cluster of tandemly repeated genes coding for P-type ion pumps. The sequence alterations relative toPMR2 are mainly located at the C-terminus of the protein.     

Linkage of a new member of the lectin supergene family to chicken Mhc genes

With the use of tissue-specific cDNA probes, several genes, which do not correspond to the class I (B-F), class II (B-L), or class IV (B-G) genes, were detected within the cosmid clusters containing the chicken major histocompatibility genes. We isolated cDNA clones with a probe corresponding to one of them, the 17.5 gene, located between two class I genes. The 17.5.3 cDNA, isolated from a chicken spleen cDNA library, encodes a 257-residue-long protein. This sequence shows significant similarity with several members of the C-type animal lectin superfamily and is probably a type II transmembrane protein. Analysis of several cDNA clones, together with Southern blot experiments, strongly suggest that this gene belongs to a multigene family, with at least some of its members being polymorphic. Several arguments lend support to the possibility that, together with the linked Mhc genes, the 17.5 gene is part of the recently described Rfp-Y system.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M88072.     

The pet genes of Rhodospirillum rubrum: cloning and sequencing of the genes for the cytochrome bc1-complex

Summary A cytochrome bc ₁-complex of Rs. rubrum was isolated and the three subunits were purified to homogeneity. The N-terminal amino acid sequence of the purified subunits was determined by automatic Edman degradation. The pet genes of Rhodospirillum rubrum coding for the three subunits of the cytochrome bc ₁-complex were isolated from a genomic library of Rs. rubrum using oligonucleotides specific for conserved regions of the subunits from other organisms and a heterologous probe derived from the genes for the complex of Rb. capsulatus. The complete nucleotide sequence of a 5500 by SalI/SphI fragment is described which includes the pet genes and three additional unidentified open reading frames. The N-terminal amino acid sequence of the isolated subunits was used for the identification of the three genes. The genes encoding the subunits are organized as follows: Rieske protein, cytochrome b, cytochrome c ₁. Comparison of the N-terminal protein sequences with the protein sequences deduced from the nucleotide sequence showed that only cytochrome c ₁ is processed during transport and assembly of the three subunits of the complex. Only the N-terminal methionine of the Rieske protein is cleaved off. The similarity of the deduced amino acid sequence of the three subunits to the corresponding subunits of other organisms is described and implications for structural features of the subunits are discussed.Abbreviations BSA bovine serum albumin - SDS sodium dodecylsulphate - Rs Rhodospirillum - Rb Rhodobacter - Pc Paracoccus - Rps Rhodopseudomonas The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X55387     

Characterisation of the <Emphasis Type="Italic">Aspergillus niger dapB</Emphasis> gene,which encodes a novel fungal type IV dipeptidyl aminopeptidase

We have cloned the Aspergillus niger dapB gene. Analysis of its nucleotide sequence and the corresponding protein sequence indicates that the gene encodes a type IV dipeptidyl aminopeptidase (DPP IV). Based upon its deduced sequence we predict the presence of a transmembrane domain in the protein. Furthermore, dapB-overexpressing transformants display an increase in intracellular DPP IV activity. This is the first reported characterisation of a dipeptidyl aminopeptidase with a transmembrane domain from a filamentous fungus. Using the dapB sequence as a query, we were able to identify 14 DPP IV-encoding genes, and 12 additional DPPIV proteases in public genomic databases. Phylogenetic analysis reveals that in yeasts there are two clades of genes that encode DPP IV proteases with a transmembrane domain. In this study we demonstrate that, as in yeasts, two classes of DPP IV-encoding genes exist in filamentous fungi. However, only one of these codes for DPP IV proteases with a transmembrane domain. The second type present in filamentous fungi encodes extracellular DPP IV proteases. The dapB gene belongs to the first cluster. We propose that DapB plays a role in the proteolytic maturation of enzymes produced by A. niger.Electronic Supplementary Material Supplementary material is available for this article at     

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12

Summary In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of M_r 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.     

Nucleotide sequence of class D tetracycline resistance genes from Salmonella ordonez

Summary Plasmid pIP173, isolated from Salmonella ordonez strain BM2000, confers resistance to tetracycline and a number of other antibiotics. We determined the nucleotide sequence of the pIP173 tetR repressor and tetA resistance genes. The pIP173 tetR gene is essentially identical to the class D tetR gene from plasmid RA1. The pIP173 tet genes are flanked by directly repeated copies of the insertion sequence IS26. Interestingly, the 3 end of the tetR gene, encoding the C-terminal 16 amino acids of the TetR protein, extends into the flanking IS26 sequence. The relationships between the class A, B, C, and D TetA sequences parallel the relationships between the corresponding TetR sequences; class D is more closely related to class B than to either class A or C. Overall, the four TetA sequences show 38% identity and 57% similarity.     

Structural and functional analysis of the fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512

The fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512 were identified by DNA hybridization of a genomic library with an internal fragment of the Rhizobium meliloti fixJ gene. The nucleotide sequence was determined and the corresponding amino acid sequence was aligned with the amino acid sequences of the FixL proteins of R. meliloti, Bradyrhizobium japonicum and Azorhizobium caulinodans. While the FixJ protein and the carboxy-terminal part of the FixL protein are highly homologous to the other FixL and FixJ proteins, the homology in the central heme-binding, oxygen-sensing domain and in the amino-terminal domain of FixL is very low. The R. leguminosarum bv. phaseoli FixL protein does not contain the heme-binding motif defined for the previously described FixL proteins. R. leguminosarum bv. phaseoli fixLJ and fixJ mutants were constructed. These mutants can still fix nitrogen, albeit at a reduced level. Expression analysis of nifA-gusA and nifH-gusA fusions in the constructed mutants revealed that the R. leguminosarum bv. phaseoli fixLJ genes are involved in microaerobic nifH expression but not in nifA expression.The nucleotide sequence data reported will appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under the accession number U27314     

Molecular cloning and analysis of CDC28 and cyclin homologues from the human fungal pathogen Candida albicans

In the budding yeast Saccharomyces cerevisiae, progress of the cell cycle beyond the major control point in G1 phase, termed START, requires activation of the evolutionarily conserved Cdc28 protein kinase by direct association with GI cyclins. We have used a conditional lethal mutation in CDC28 of S. cerevisiae to clone a functional homologue from the human fungal pathogen Candida albicans. The protein sequence, deduced from the nucleotide sequence, is 79% identical to that of S. cerevisiae Cdc28 and as such is the most closely related protein yet identified. We have also isolated from C. albicans two genes encoding putative G1 cyclins, by their ability to rescue a conditional GI cyclin defect in S. cerevisiae; one of these genes encodes a protein of 697 amino acids and is identical to the product of the previously described CCN1 gene. The second gene codes for a protein of 465 residues, which has significant homology to S. cerevisiae Cln3. These data suggest that the events and regulatory mechanisms operating at START are highly conserved between these two organisms.     

Cloning and characterization of a secY homolog from Chlamydia trachomatis

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc- ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50 195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SceY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.     

Drought,heat and salt stress induce the expression of a citrus homologue of an atypical late-embryogenesis Lea5 gene

In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated. Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character. The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension. The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl. This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress. We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5.     

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins

Summary The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.