Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7

The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.     

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.     

Autoregulation of the pTF-FC2 proteic poison-antidote plasmid addiction system (pas) is essential for plasmid stabilization

The stabilization of a test plasmid by the proteic, poison-antidote plasmid addiction system (pas) of plasmid pTF-FC2 was host strain dependent, with a 100-fold increase in stability in Escherichia coli CSH50, a 2.5-fold increase in E. coli JM105, and no detectable stabilization in E. coli strains JM107 and JM109. The lethality of the PasB toxin was far higher in the E. coli strains in which the pas was most effective. Models for the way in which poison-antidote systems stabilize plasmids require that the antidote have a much higher rate of turnover than that of the toxin. A decrease in host cell death following plasmid loss from an E. coli lon mutant and a decrease in plasmid stability suggested that the Lon protease plays a role in the rate of turnover of PasA antidote.     

Rates of spontaneous mutation in an archaeon from geothermal environments

To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 +/- 0.7) x 10(-7) and (1.5 +/- 0.6) x 10(-7) mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37 degrees C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells.     

Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon

The gene encoding an extremely stable inorganic pyrophosphatase from Sulfolobus sp. strain 7, a thermoacidophilic archaeon, was cloned and sequenced. An open reading frame consisted of 516 base pairs coding for a protein of 172-amino acid residues. The deduced sequence was supported by partial amino acid sequence analyses. All the catalytically important residues were conserved. A unique 17-base-pair sequence motif was found to be repeated four times in frame in the gene, encoding a cluster of acidic amino acids essential for the function. Although the codon usage of the gene was quite different from that of Escherichia coli, the gene was effectively expressed in E. coli. Coexpression of tRNA(Arg), cognate for the rare codon AGA in E. coli, however, further improved the production of the enzyme, which occupied more than 85% of the soluble proteins obtained after removal of heat denatured E. coli proteins.     

Hin-mediated inversion on positively supercoiled DNA

Hin recombinase requires negatively supercoiled DNA for an efficient inversion. We have generated positively supercoiled plasmid DNA using reverse gyrase from Sulfolobus shibatae and subjected it to the Hin-mediated inversion reaction. Both Hin and Fis showed the same DNA binding activity regardless of the superhelical handedness of the substrate plasmid. However, inversion activity on positively supercoiled DNA was less than 1% of negatively supercoiled DNA. Assays designed to probe steps in inversion, showed that on positively supercoiled DNA, Hin was able to cleave the recombination sites with the same efficiency shown on negatively supercoiled DNA but was not able to exchange the cleaved DNA. Based on the theoretical differences between positive and negative supercoiling, our data may suggest that unwinding of the double helix at recombination sites is needed after DNA cleavage for strand exchange to occur.     

Expression of the Solfolobus acidocaldarius Rieske iron sulfur protein II (SOXF) with the correctly inserted [2FE-2S] cluster in Escherichia coli

The Rieske protein II (Schmidt et al., 1996, FEBS Lett. 388, 43-46) from the thermoacidophilic crenarcheon Sulfolobus acidocaldarius (DSM 639) was expressed in E. coli cells. The full length protein was strictly bound to the E. coli membranes and could only be removed by detergent treatment indicating the presence of a membrane anchor. The iron sulfur cluster was correctly inserted into a fraction of the full length protein and much more effectively into a soluble form created by the deletion of the 45 N-terminal amino acids. The soluble form of the protein displayed the typical spectroscopic properties of a respiratory Rieske protein. The midpoint potential was +375 mV determined by CD redox potentiometry. The presented data demonstrate that the structure of the recombinant protein is very similar or identical to the authentic protein making this a powerful model system for the studies of Rieske proteins by site directed mutagenesis.     

Overexpression of the robA gene increases organic solvent tolerance and multiple antibiotic and heavy metal ion resistance in Escherichia coli

Escherichia coli K-12 OST3410 was isolated previously as a stable cyclohexane-tolerant mutant derived from cyclohexane-sensitive strain JA300. A plasmid which provides cyclohexane tolerance to strain JA300 was isolated from the OST3410 genomic library. Subcloning and sequence analysis showed that the plasmid contained the robA gene, whose gene product was reported to bind specifically to the right border of oriC. We observed that the robA gene on the multicopy plasmid generally increased the organic solvent tolerance of several E. coli strains. We also observed an increase in the organic solvent tolerance of JA300 carrying the lac-robA fusion gene on a low-copy plasmid by isopropyl-beta-D-thiogalactopyranoside induction. Strain JA300 carrying the multicopy robA plasmid also showed an increase in resistance to a number of unrelated antibiotics and heavy metal ions, and the spectrum of resistance was significantly similar to that of the soxS-overexpressing strain.     

Two-minute miniprep method for plasmid DNA isolation

An extremely rapid method, INSTA-PREP, has been developed to prepare plasmid DNA from 1 to 3 mL miniprep Escherichia coli bacterial cultures. Direct extraction of plasmid DNA from E. coli bacterial cells is achieved by a two-phase solution consisting of phenol-chloroform-isoamyl alcohol and water or buffer with efficient separation of the phases by centrifugation in the presence of the INSTA-PREP gel barrier material. Processing time, from E. coli culture to usable plasmid DNA, is two minutes or less per sample. Supercoiled plasmid DNA yields ranged from 3 to 10 micrograms per mL of culture depending on plasmid copy number. Plasmid DNAs prepared by INSTA-PREP were analyzed and are suitable for use in molecular biology procedures including restriction digestion, ligation with T4 DNA ligase, bacterial transformation, PCR, cultured cell transfection and T7 DNA polymerase or thermostable DNA polymerase-mediated dideoxynucleotide sequencing.     

Biochemical and phylogenetic characterization of the dUTPase from the archaeal virus SIRV

The derived amino acid sequence from a 474-base pair open reading frame in the genome of the Sulfolobus islandicus rod-shaped virus SIRV shows striking similarity to bacterial dCTP deaminases and to dUTPases from eukaryotes, bacteria, Poxviridae, and Retroviridae. The putative gene was expressed in Escherichia coli, and dUTPase activity of the recombinant enzyme was demonstrated by hydrolysis of dUTP to dUMP. Deamination of dCTP by the enzyme was not detected. Phylogenetic analysis based on amino acid sequences of the characterized enzyme and its homologues showed that the dUTPase-encoding dut genes and the dCTP deaminase-encoding dcd genes constitute a paralogous gene family. This report is the first identification and functional characterization of an archaeal dUTPase and the first phylogeny derived for the dcd-dut gene family.     

Cloning and expression of a plasmid pBS195 gene, determining oxygenase activity, in Escherichia coli cells

Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated from long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms [1]. Plasmid-harboring colonies of the strain Escherichia coli HB101 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are present in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not bee detected in any known metapyrocatechases, were found in cells of E. coli. Hybridization with a 32P-labeled fragment containing the NahC gene revealed a region of homology with a 1.6-kb EcoR I- BamH I fragment of plasmid pBS195. Deletion variants of this plasmid that lost oxygenase activity confirmed the location of the oxygenase gene in this region. The gene responsible for oxygenase activity in the plasmid was cloned on the pUC19 vector in E. coli cells. The expression of the cloned gene is controlled by the lac promoter of this vector. Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synthesized in E. coli mini-cells, showed that this activity requires the participation of a polypeptide with molecular mass of 34 kDa.     

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.     

A family of stability determinants in pathogenic bacteria

A novel segregational stability system was identified on plasmid R485, which originates from Morganella morganii. The system is composed of two overlapping genes, stbD and stbE, which potentially encode proteins of 83 and 93 amino acids, respectively. Homologs of the stbDE genes were identified on the enterotoxigenic plasmid P307 from Escherichia coli and on the chromosomes of Vibrio cholerae and Haemophilus influenzae biogroup aegyptius. The former two homologs also promote plasmid stability in E. coli. Furthermore, the stbDE genes share homology with components of the relBEF operon and with the dnaT gene of E. coli. The organization of the stbDE cassette is reminiscent of toxin-antitoxin stability cassettes.     

Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Cloned genes of the aerobactin system of virulent avian Escherichia coli do not confer virulence to recombinant strains

1. We cloned the aerobactin region and its receptor from pMV14, a large nonconjugative plasmid isolated from the virulent strain UEL14, to assess the importance of the aerobactin iron uptake system as a virulence determinant in septicemic avian Escherichia coli. 2. The physical map of the region of the recombinant plasmid (pGMV1) containing the genes for synthesis of aerobactin and its receptor was very similar to the corresponding region in pABN1 containing the genetic determinants for the aerobactin system of pColV-K30. 3. The 74-kDa outer-membrane protein encoded by pGMV1 cross-reacted immunologically with the 74-kDa aerobactin receptor protein encoded by pABN1. 4. Various avirulent E. coli strains carrying the recombinant plasmid, which contains only the aerobactin system, were assayed for virulence and were found to be avirulent for chickens. Only the wild-type aerobactin-producing strain was virulent in a pathogenicity test for chickens. 5. These results show that the aerobactin system by itself does not confer virulence, and that other factors are necessary for virulence of avian strains of E. coli.     

Plasmid DNA strand breaks induced by laser irradiation of 193 nm wavelength

DNA of plasmid pBR322 irradiated with laser at a wavelength of 193 mm was treated with an extract containing proteins from E.coli K12 AB1157 (wild-type). The enzymes were found to produce single- and double-strand DNA breaks, which was interpreted as a transformation of a portion of cyclobutane pyrimidine dimers and (6-4) photoproducts into nonrepairable single-strand DNA breaks. The products resulted from ionization of DNA, in particular, single-strand breaks, transform to double-strand breaks. A comparison of these data with the data on survival of plasmid upon transformation of E.coli K12 AB1157 enables one to assess the biological significance of single- and double-strand breaks. The inactivation of the plasmid (in AB1157) is mainly determined by the number of directly formed laser-induced single-strand breaks, whereas the contribution of enzymatically produced single- and double-strand breaks is insignificant.     

Effect of duodenum-preserving resection of the head of the pancreas on endocrine and exocrine pancreatic function in patients with chronic pancreatitis

Two mobilizable cloning vectors, designated pABW1 and pAWB2, were constructed basing on the E. coli vector pBGS18 and oriT originating from RK2. In pABW2 the kanamycin resistance gene was replaced by a novel tetracycline resistance cassette derived from Tn1721. Both vectors, specific for E. coli, allow to perform the cloning steps in E. coli and then to efficiently transfer the constructs by conjugation to the host of choice. A vector which cannot propagate in the given host can be applied for identification of the host specific plasmid replicator regions. With the use of pABW2 we defined the minimal replicator region of pTAV202-a mini-derivative of the large pTAV1 plasmid of P. versutus. We also proved that RepC' encoded on this fragment is the principal initiator replication protein and that oriV is located along its coding sequence.     

Trans activation of the Escherichia coli ato structural genes by a regulatory protein from Bacillus megaterium: potential use in polyhydroxyalkanoate production

A Bacillus megaterium genomic fragment, which encoded an activator homologous to sigma 54 regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B. megaterium screened for beta-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E. coli strains.     

16S rRNA mutation associated with tetracycline resistance in a gram-positive bacterium

A genetic basis for tetracycline resistance in cutaneous propionibacteria was suggested by comparing the nucleotide sequences of the 16S rRNA genes from 16 susceptible and 21 resistant clinical isolates and 6 laboratory-selected tetracycline-resistant mutants of a susceptible strain. Fifteen clinical isolates resistant to tetracycline were found to have cytosine instead of guanine at a position cognate with Escherichia coli 16S rRNA base 1058 in a region important for peptide chain termination and translational accuracy known as helix 34. Cytosine at base 1058 was not detected in the laboratory mutants or the tetracycline-susceptible strains. The apparent mutation was recreated by site-directed mutagenesis in the cloned E. coli ribosomal operon, rrnB, encoded by pKK3535.E. coli strains carrying the mutant plasmid were more resistant to tetracycline than those carrying the wild-type plasmid both in MIC determinations and when grown in tetracycline-containing liquid medium. These data are consistent with a role for the single 16S rRNA base mutation in clinical tetracycline resistance in cutaneous propionibacteria.