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PubMed1994

Cloning, sequencing, and viscometric adhesion analysis of heat-resistant agglutinin 1, an integral membrane hemagglutinin from Escherichia coli O9:H10:K99.

The gene encoding a mannose-resistant hemagglutinating protein was cloned from Escherichia coli O9:H10:K99. The hemagglutinin is different from two other mannose-resistant hemagglutinins in this strain, K99 and F41. The agglutinin, named heat-resistant agglutinin 1 (HRA1) since heating to 70 degrees C does not destroy its aggregative properties, strongly agglutinates human, pig, and dog erythrocytes, shows little or no affinity towards cow and chicken erythrocytes, but agglutinates human colon adenocarcinoma 201 (COLO 201) cells. The hra1 gene present on the recombinant plasmid pETE1 was localized by subcloning, and its nucleotide sequence was determined. The gene consists of a 792-bp open reading frame coding for a putative protein of 29 kDa with a predicted N-terminal secretory signal sequence. HRA1 shares no significant identity with data base protein sequences. HRA1 is strongly associated with the bacterial membrane, resisting sonication and isolation attempts based upon standard adhesin purification techniques. N-terminal sequencing of a unique 25-kDa band present in polyacrylamide gels of outer membrane preparations of bacteria harboring pETE1 correlated with the predicted N-terminal amino acid sequence of HRA1 after cleavage of the signal peptide. A viscometric agglutination assay sensitive to the strength of bacterial adhesion shows that the agglutination mediated by bacteria expressing HRA1 is weaker than that of bacteria bearing the F41 adhesin, probably because of the high-molecular-weight, multivalent nature of the latter adhesin. Our observations suggest that HRA1 is a monomeric outer membrane agglutinin.

Adhesins, Bacterial↗

PubMed1997

Functional analysis of the Shigella flexneri IpaC invasin by insertional mutagenesis.

The ability of Shigella to enter epithelial cells, to escape from the phagocytic vacuole, and to induce apoptosis in macrophages requires the IpaB, IpaC, and IpaD proteins. An extracellular complex containing IpaB and IpaC can promote the uptake of inert particles by epithelial cells. To determine whether the function of IpaC is to act as an extracellular chaperone for IpaB in the Ipa complex or as an effector of entry involved in a direct interaction with the cell surface, we have constructed eight IpaC recombinant proteins by inserting the coding sequence for a 12- to 14-amino-acid fragment into restriction sites scattered within the ipaC gene. We have investigated the ability of recombinant proteins to bind IpgC in the bacterial cytoplasm and IpaB in the extracellular medium and to complement an ipaC null mutant for entry into HeLa cells, lysis of erythrocytes, and escape from the phagocytic vacuole in infected macrophages. Most recombinant proteins were produced and secreted at a level similar to that of wild-type IpaC and did not exhibit altered susceptibility to proteolysis by trypsin, and all were able to bind IpgC and IpaB. Some recombinant proteins did not complement the ipaC mutant for entry into HeLa cells, lysis of erythrocytes, or escape from the phagocytic vacuole, which indicates that IpaC plays an active role in these processes and does not act solely as a chaperone for IpaB. In addition, some insertions which were located outside of the hydrophobic region of IpaC differentially affected the abilities of Shigella to enter epithelial cells and to lyse cell membranes.

Antibodies, Monoclonal↗

PubMed1995

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene.

Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5. Expression of the P5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum bv. viciae UPM791 gene bank by complementing an R. meliloti FixK- mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN- mutant of R. leguminosarum bv. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P(5b) was reduced to ca. 50% of the wild-type level in the FnrN(-) mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum by. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.

Aerobiosis↗

PubMed2001

Alternative bacteriophage display systems.

Filamentous phage has been extensively used to implement various aspects of phage display technology. The success of these organisms as vectors to present foreign peptides and to link them to their coding sequences is a consequence of their structural and biological characteristics. Some of these properties, however, represent a limitation when one attempts to display proteins that cannot be efficiently exported through the bacterial membrane or do not fold properly in the periplasm. Thus, the desirability of developing alternative display systems was recognised recently and led to the development of a different class of display vectors that assemble their capsid in the cytoplasm and are released via cell lysis. This review describes and compares the properties of these alternative display systems.

Bacteriophages↗

PubMed1994

Molecular cloning of gyrA and gyrB genes of Mycobacterium tuberculosis: analysis of nucleotide sequence.

We have recently reported the cloning of gyrA and gyrB genes from Mycobacterium tuberculosis H37Ra [Curr. Science, (1994) 66, 664-667]. Here, we present the complete nucleotide sequence of gyrB gene from M. tuberculosis H37Ra along with the flanking regions. The gyrA gene has been located 34 nucleotides downstream of gyrB and has been partially sequenced; both the genes seem to be transcribed from the promoter elements located upstream of gyrB coding sequence. The gyrB gene encodes a polypeptide of 714 amino acids. The deduced amino acid sequences of gyrB and a part of gyrA show extensive homology to the corresponding genes from other bacterial species. The DNA gyrase of M. tuberculosis could be utilised to develop new line of antitubercular drugs.

Amino Acid Sequence↗

PubMed1993

Sequences of tRNA-encoding genes and associated open reading frames of Streptomyces lividans.

A gene library of Streptomyces lividans has been screened for tRNA-encoding genes with labeled Streptomyces tRNA as a probe. By sequence analysis of hybridizing fragments, two single genes have been identified which code for tRNA(Asp) and tRNA(Gly). Associated with the tRNA(Gly) gene, there are three open reading frames (ORFs) which might code for gene products possibly involved in active transport processes through the bacterial membrane. The transcriptional organization of tRNAGly and the following ORFs was examined by high-resolution S1 mapping. A third clone carried a cluster of genes which encode two tRNA(Gln) and three tRNA(Glu). This cluster corresponds to a similar cluster previously described for Streptomyces rimosus [Plohl and Gamulin, Mol. Gen. Genet. 222 (1990) 129-134].

Amino Acid Sequence↗

PubMed2003

Optimized bacterial expression of human apolipoprotein A-I.

Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100+/-20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp-Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product protein's primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.

Apolipoprotein A-I↗

PubMed2006

SIDDBASE: a database containing the stress-induced DNA duplex destabilization (SIDD) profiles of complete microbial genomes.

Prokaryotic genomic DNA is generally negatively supercoiled in vivo. Many regulatory processes, including the initiation of transcription, are known to depend on the superhelical state of the DNA substrate. The stresses induced within DNA by negative superhelicity can destabilize the DNA duplex at specific sites. Various experiments have either shown or suggested that stress-induced DNA duplex destabilization (SIDD) is involved in specific regulatory mechanisms governing a variety of biological processes. We have developed methods to evaluate the SIDD properties of DNA sequences, including complete chromosomes. This analysis predicts the locations where the duplex becomes destabilized under superhelical stress. Previous studies have shown that the SIDD-susceptible sites predicted in this way occur at rates much higher than expected at random in transcriptional regulatory regions, and much lower than expected in coding regions. Analysis of the SIDD profiles of 42 bacterial genomes chosen for their diversity confirms this pattern. Predictions of SIDD sites have been used to identify potential genomic regulatory regions, and suggest both possible regulatory mechanisms involving stress-induced destabilization and experimental tests of these mechanisms. Here we describe the SIDDBASE database which enables users to retrieve and visualize the results of SIDD analyses of completely sequenced prokaryotic and archaeal genomes, together with their annotations. SIDDBASE is available at www.gc.ucdavis.edu/benham/siddbase.

DNA, Archaeal↗

PubMed1994

Cloning and expression of a soluble sialidase from Chinese hamster ovary cells: sequence alignment similarities to bacterial sialidases.

A cDNA encoding a soluble sialidase from Chinese hamster ovary (CHO) cells has been cloned and expressed. Completely degenerate oligonucleotide primers, which were based on the amino acid sequence of peptides obtained from the purified sialidase (Warner et al., Glycobiology, 3, 455-463, 1993), and the polymerase chain reaction, with single-stranded cDNA template, were employed to generate a unique oligonucleotide probe. The unique probe of 93 bp was used for screening a lambda gt 10 CHO cell cDNA library. A single clone, which contained a 1.4 kb insert, was isolated after screening 450,000 recombinants. The complete coding region of the protein, 1137 nucleotides, was contained in the isolated clone and it predicted a protein of 379 amino acids. The insert had a 186 bp 5' non-coding leader sequence and a 40 bp 3' non-coding region. No signal peptide was identified in the insert, suggesting a cytosolic localization for the protein. No significant primary sequence identities were observed when the deduced amino acid sequence of the CHO cell sialidase was compared with other mammalian proteins or microbial sialidases. However, the protein had significant sequence alignment similarity with several bacterial sialidases. Two 'Asp box' motifs in the CHO cell sialidase had a remarkable alignment positioning in the protein sequence with the similar motifs of the Salmonella LT2 and Clostridium perfringens sialidases. High levels of the enzyme were expressed in Spodoptera frugiperda cells infected with a modified Autographa californica nuclear polyhedrosis virus harbouring the sialidase cDNA.

Amino Acid Sequence↗

PubMed1990

Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum.

Arabidopsis Proteins↗

PubMed1997

Primary structure of an invertebrate dihydrolipoamide dehydrogenase with phylogenetic relationship to vertebrate and bacterial disulfide oxidoreductases.

Dihydrolipoamide dehydrogenase (E3) is a flavoprotein component of multi-enzyme complexes catalyzing oxidative decarboxylation of alpha-ketoacids in the Krebs' cycle. We have cloned a 2.4-kb E3 cDNA from an arthropod, Manduca sexta, that codes for 497 amino acids and translates to a 51-kDa protein in vitro. Sequences at and around the dinucleotide binding domains, disulfide active site and the C-terminal interface domain involved in substrate binding are highly conserved in Manduca E3. Phylogenetic analysis of protein sequences from the flavoprotein class of disulfide oxidoreductases family of enzymes suggests that in spite of the homologous nature of E3 and glutathione reductase (goR) in sequence and structure, E3 shares a common ancestor with mercuric reductase (merA), whereas goR is more related to trypanothione reductase (tryR) than to other members. All members, except goRs, seemed to be monophyletic. Plant goRs seemed to have arisen differently and are more closely related to tryRs than to bacterial and vertebrate goRs. Earlier speculation on the nature of origin of E3 in Pseudomonas is not supported by phylogenetic data. A possible structural relationship of Manduca E3 to other pyridine-binding proteins, such as the neurotransmitter transporters and channels, is proposed.

Amino Acid Sequence↗

PubMed2000

estA, a gene coding for a cell-bound esterase from Paenibacillus sp. BP-23, is a new member of the bacterial subclass of type B carboxylesterases.

Screening of a gene library from Paenibacillus sp. BP-23 generated in Escherichia coli led to identification of a clone that directed the production of lipolytic activity. From the sequencing data, we found an open reading frame encoding a protein of 485 amino acids with an estimated molecular mass of 53 kDa and a pI of 5.1. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-XI-S-X2-G included in most serine-esterases and lipases. The cloned protein showed high homology with enzymes belonging to the bacterial subclass of type B carboxylesterases. The enzyme had a significant preference for esters of short-chain fatty acids and showed the kinetics behaviour of a true esterase. Maximum activity was found at pH 7.5 and 37 degrees C, although the enzyme was active in the pH range 6.0- 9.0 and at temperatures up to 45 degrees C. As expected for a serine-esterase, activity was inhibited by phenylmethylsulphonyl fluoride.

Amino Acid Sequence↗

PubMed2000

Characterization of a cDNA encoding a rice mitochondrial voltage-dependent anion channel and its gene expression studied upon plant development and osmotic stress.

The voltage-dependent anion channel (VDAC) of mitochondria forms a large pore in the outer envelope membrane. Here, the full Oryza sativa OSVDAC1 cDNA was sequenced and is shown to belong to a small multigene family in the rice genome. This cDNA is 1093 bp long and codes for a protein of 274 amino acids. Expression studies of the osvdac1 gene show a regulation of its level in function of the plantlets maturation and organs. In contrast with several bacterial porins, osmotic stress does not have any effect on the plant osvdac1 gene expression.

Amino Acid Sequence↗

PubMedDate not supplied

[Structure and evolutionary role of the Penelope mobile element in Drosophila species of the virilis group].

The mobile element Penelope is activated and mobilizes several other transposons in dysgenic crosses in Drosophila virilis. Its structure proved to be complex and to vary greatly in all examined species of the virilis group. Phylogenetic analysis of the reverse transcriptase (RT) domain assigned Penelope to a new branch, rather than to any known family, of LTR-lacking retroelements. Amino acid sequence analysis showed that the C-terminal domain of the Penelope polyprotein is an active endonuclease, which is related to intron-encoded endonucleases and to bacterial repair endonuclease UrvC, and may act as an integras. Retroelements coding for a putative endonuclease that differs from typical integrase have thus far not been known. The N-terminal domain of the Penelope polyprotein was shown to contain a protease with significant homology to HIV-1 protease. Phylogenetic analysis divided the Penelope copies from several virilis species into two subfamilies, one including virtually identical full-length copies, and the other comprising highly divergent defective copies. The results suggest both vertical and horizontal transfer of the element. Possibly, Penelope invasion recurred during evolution and contributed to genome rearrangement in the virilis species. Chromosome aberrations detected in D. virilis, which is now being invaded by Penelope, is direct evidence for this assumption.

Alcohol Dehydrogenase↗

PubMed1995

Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae.

The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox-active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N-terminal part of the protein corresponds to TR and the C-terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.

Amino Acid Sequence↗

PubMed1991

Isolation and sequence of a cDNA encoding mouse IMP dehydrogenase.

Inosinic acid (IMP) dehydrogenase (IMPD) catalyzes the conversion of IMP to XMP as the first committed step in GMP biosynthesis de novo. We have isolated a cDNA containing the complete coding region of mouse IMPD by its ability to complement a bacterial mutant lacking IMPD activity. Two independent cDNA clones were isolated by complementation, of which the longest was 1.7 kb in length. Northern analyses, using the IMPD cDNA as a probe, indicated that mature IMPD mRNA was a single species approx. 2.0 kb in size. Mouse IMPD is almost identical to Chinese hamster and human IMPDs and is highly conserved between Escherichia coli and mouse, with a direct amino acid (aa) identity of 39%, which increases to 60% if conserved aa are considered. The leader region of our longest cDNA clone is G + C-rich and contains two tandem copies of a G + C-rich direct repeat.

Amino Acid Sequence↗

PubMed1987

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains.

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.

Bacillus thuringiensis↗

PubMed2000

Distribution of hammerhead and hammerhead-like RNA motifs through the GenBank.

Hammerhead ribozymes previously were found in satellite RNAs from plant viroids and in repetitive DNA from certain species of newts and schistosomes. To determine if this catalytic RNA motif has a wider distribution, we decided to scrutinize the GenBank database for RNAs that contain hammerhead or hammerhead-like motifs. The search shows a widespread distribution of this kind of RNA motif in different sequences suggesting that they might have a more general role in RNA biology. The frequency of the hammerhead motif is half of that expected from a random distribution, but this fact comes from the low CpG representation in vertebrate sequences and the bias of the GenBank for those sequences. Intriguing motifs include those found in several families of repetitive sequences, in the satellite RNA from the carrot red leaf luteovirus, in plant viruses like the spinach latent virus and the elm mottle virus, in animal viruses like the hepatitis E virus and the caprine encephalitis virus, and in mRNAs such as those coding for cytochrome P450 oxidoreductase in the rat and the hamster.

Amino Acid Motifs↗