Multicopy plasmid suppression of stationary phase chaperone toxicity in Escherichia coli by phosphogluconate dehydratase and the N-terminus of DnaK

Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect. This effect is most acute in stationary phase cells. A selection scheme was developed to isolate multicopy suppressors from an E. coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK. Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively. Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors. DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK′). Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK′) as determined by gel electrophoresis. Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins. Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors. These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.     

Photosensory transduction in Euglena gracilis: Effect of some metabolic drugs on the photophobic response

We have investigated the effect of some metabolic drugs, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,4-dinitrophenol (DNP), sodium azide (NaN₃), on the photobehavior of single cells of Euglena gracilis, in order to clarify the relevance of different metabolic pathways in the process of photoperception and sensory transduction in this alga. The results obtained show that the photophobic response of Euglena is not affected by the action of these drugs. This suggests that neither the photosynthetic process nor oxidative phosphorylation play a significant role in the phenomenon of photosensory transduction in Euglena.List of Abbreviations DNP 2,4-dinitrophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI Photosystem I - PSII Photosystem II     

Transient suppression of F-plasmid incompatibility in a strain of Escherichia coli

Summary The incompatibility between F plasmids is transiently suppressed in Escherichia coli strain CSH54. As a result this strain is able to maintain two F factors or an F factor and a mini-F plasmid for considerably longer periods than normal strains. When selective pressure for two markers carried by two separate Fs (or an F and mini-F) is imposed on normal strains, the two plasmids tend to form a cointegrate structure which can be detected genetically by the joint transfer of both the markers upon mating. This does not happen in CSH54; instead, the two plasmids are maintained and transferred independently. Physical evidence for the maintenance of an F and a mini-F plasmid is provided by agarose gel electrophoresis.     

Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis.

Summary During conjugative transfer of sex pheromone plasmids ofEnterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. We report here the DNA sequence of a 3 .8 kb fragment of the sex pheromone plasmid pAD1 containing the structural genesea1 for surface exclusion protein and a small open reading frame (ORF) upstream ofsea1. Surface exclusion protein Seal was found to be highly homologous to the surface exclusion protein Sec10 encoded by the sex pheromone plasmid pCF10. Hybridization studies with DNA probes derived from the structural gene seal demonstrated that, with the exception of pAM373, all known sex pheromone plasmids carry a homologous gene. These studies also indicated that the genetic organization is similar in these plasmids, with the structural gene for surface exclusion protein being located 5 to that for aggregation substance.     

Replication properties ofARS1 plasmids inSaccharomyces cerevisiae: dependence on the carbon source

The replication behaviour of a number ofARS1-based plasmids was investigated on propagation inSaccharomyces cerevisiae grown with either glucose or galactose as carbon source. Growth on galactose results in reduced plasmid stability, as well as in reduced replication efficiency, when the entire 1.5-kbTRP1-ARS1 fragment is present on a plasmid. The galactose sensitivity is mediated by a 0.13-kb fragment harbouring part of theGAL3 promoter. This fragment exerts its effect when situated either 5 or 3 to the ARS core consensus at distances up to 0.9 kb. The endogenous 2 µm plasmid remained unaffected by the choice of carbon source.     

Linear plasmid DNAs of the plant pathogenic fungus Rhizoctonia solani with unique terminal structures

Summary Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3-exonuclease and 5-exonuclease even after treatment with proteinase K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of the plasmids is discussed.     

Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion

Summary The Escherichia coli mutator gene mutT, which causes A:TC:G transversion, was cloned in pBR 322. mutT ⁺ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E. coli chromosome. Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons. The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid. The nucleotide sequence of the mutT gene was determined by the dideoxy method.Abbreviations Ap ampicillin - IPTG isopropyl--d-thiogalactopyranoside - kb kilobase pair(s) - kDa kilodalton(s) - SDS sodium dodecyl sulphate - Tc tetracycline     

Identification of the domain of Saccharomyces cerevisiae adenylate cyclase associated with the regulatory function of RAS products

Summary Various truncated CYR1 genes of Saccharomyces cerevisiae were fused to efficient promoters and expressed in Escherichia coli and S. cerevisiae cells with or without the RAS genes. The catalytic domain of adenylate cyclase encoded by the 3-terminal 1.3 kb region of the open reading frame of the CYR1 gene produced cyclic AMP, irrespective of the presence of RAS genes. The product of the 3-terminal 2.1 kb region of CYR1 showed guanine nucleotidedependent adenylate cyclase activity and produced a large amount of cAMP in the presence of the RAS gene. Thus, the domain encoded by the 0.8 kb region adjacent to the catalytic domain is associated with the regulatory function of the RAS products. The cyr1 RAS1 RAS2 cells carrying the 3-terminal 1.3 kb region of CYR1 were unable to respond to environmental signals such as sulfur starvation and temperature shift, but the cyr1 cells carrying the 2.1 kb region and at least one RAS gene were able to respond to these signals. The environmental signals may be transferred to the adenylate cyclase system through the RAS products.     

The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression

Summary The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal c. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5 and 3 flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103154) which shows 34% similarity with the respective E. coli enzyme. Present address: Institut für Biotechnologie 1 der Kernforschungsanlage, Postfach 1913, D-5170 Jülich, Federal Republic of Germany     

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5 ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.     

Comparative analysis of 18 sex pheromone plasmids fromEnterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3-terminus of the surface exclusion gene,sep1, of sex pheromone plasmid pPD1 inEnterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.     

Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure

The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped. Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A. vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58. The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1 % homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype Il strain from wild cherry. The 3 non-coding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 by fragment derived from the coding sequence of an ipt gene of unknown origin. A comparison of different ipt gene sequences indicates that the corresponding 62 by sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 by sequence in the 3 non-coding region. In pTi82.139 the original coding region of the ipt gene has remained largely unmodified. The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 by repeat in the 3 part of the coding sequence. This leads to the loss of four glutamic acid residues from a series of ten. In spite of these differences, the ipt and 6b genes of pTiAB4 are functional. Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements. Possible implications for the study of bacterial phylogeny are discussed.     

The bacteriophage T4 dexA gene: sequence and analysis of a gene conditionally required for DNA replication

Summary We have cloned and sequenced a bacteriophage T4 EcoRI fragment that complements T4 del (39-56) infections of an optA defective Escherichia coli strain. Bacteria containing this recombinant plasmid synthesize two new proteins with molecular weights of 9 and 26 kilodaltons. We have identified the gene encoding the 26 kilodalton protein as essential for T4 infections of optA defective E. coli. Genetic and biochemical results are consistent with the identification of this protein as the product of the dexA gene, which encodes a 3 to 5 exonuclease.     

Characterization of the genes and attachment sites for site-specific integration of plasmid pSE101 in Saccharopolyspora erythraea and Streptomyces lividans

The 11.3 kb plasmid pSE101 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site and into the chromosome of Streptomyces lividans at many sites. Multisite integration in S. lividans was also observed when a 1.9 kb segment of pSE101 containing attP and adjacent plasmid sequence was used to transform a pSE101^– S. lividans host. Nucleotide sequencing of this segment revealed the presence of a complete open reading frame (ORF) designated int, encoding a putative polypeptide of 448 amino acids that shows similarities to site-specific recombinases of the integrase family. Sequencing of the 1.3 kb segment upstream of int revealed the presence of three additional ORFs: the one most distal to int encodes a putative 76 amino acid basic polypeptide analogous to the Xis proteins of a number of bacteriophages. Nucleotide sequencing of attP, and the attB, attL and attR sites from Sac. erythraea revealed a 46 by sequence common to all sites with no duplications of chromosomal sequences in the integrated state. A putative structural gene for a tRNA^Thr was found to overlap the 46 by common sequence at attB. Sequencing of four pSE101 integration sites (attB) and corresponding attL and attR sites in S. lividans showed that the 46 by sequence was present at each attR site, whereas only the first three bases, CTT, were retained at each attL and attB site. A feature common to the four attB sites and to attB is a highly conserved 21 by segment with inverted repeats flanking the CTT sequence. This indicates that crossover at each attB site in S. lividans employed attP and a site within a 5 by sequence in attB and suggests that the secondary structure of the 21 by sequence is important for site-specific integration at attB or attB.     

Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121

We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extract. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5-G/GNCC-3) in this site to generate a three base 5 overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10³ viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.     

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Summary Previous genetic analyses indicated that translational frameshifting in the –1 direction occurs within the run of six adenines in the sequence 5-TTAAAAAACTC-3 at nucleotide positions 305–315 in IS 1, where the two out-of-phase reading frames insA and B-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84–87. IS 1 mutants with a 1 by insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84–87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-LysLys-Leu. An IS 1 mutant with the DNA segment 5-CTTAAAAACTC-3 at positions 305–315 carrying the termination codon TAA in the B-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The -galactosidase activity specified by several IS 1- lacZ fusion plasmids, in which B-insB is in-frame with lacZ, showed that the region 292–377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu - Lys - Lys - Leu encoded by the DNA segment 5-TTAAAAAACTC-3, indicating that –1 frameshifting does occur within the run of adenines.     

ColE plasmid replication in DNA polymerase I-deficient strains ofEscherichia coli

Summary Replication of the non-conjugative plasmids ColE1, ColE2 and ColE3 has been examined in a number of DNA polymerase I-deficient strains, two of which contain the amber mutationpolA1 along with either of two temperature-sensitivesupF amber suppressors. These latter two strains produce reduced amounts of DNA polymerase I polymerizing activity of similar, if not identical properties to that produced bypolA⁺ strains. Our results indicate that the ColE plasmids require different amounts of DNA polymerase I for stable plasmid maintenance. Moreover whereas all three plasmids are maintained in a strain defective in the 53 exonuclease activity of DNA polymerase I, ColE2 and ColE3 are not stably maintained between 30° and 43° in a number of DNA polymerase I-deficient strains that are temperature-sensitive for ColE1 replication.     

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis

Summary The ptsG gene of Bacillus subtilis encodes Enzyme II^G1c of the phosphoenolpyruvate: glucose phosphotransferase system. The 3 end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III^Glc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5 end. These results, combined with the sequence of the 3 end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II^Glc and II^Nag of E. coli but which are present in the II^G1c-like protein encoded by the E. coli malX gene.     

Excision and transposition of two Ds transposons from the bronze mutable 4 derivative 6856 allele of Zea mays L

Summary The regulatory mutation bronze mutable 4 Derivative 6856 (bz-m4 D6856) contains a complex 6.7 kb Dissociation (Ds) element tagged with a duplication of low copy bz 3 flanking sequences (Klein et al. 1988). This creates a unique opportunity to study the transposition of a single member of the repetitive family of Ds elements. Eighteen full purple revertants (Bz alleles) of bz-m4 were characterized enzymatically and by genomic mapping. For 17 of the Bz alleles, reversion to a wild-type phenotype was caused by excision of the 6.7 kb Ds transposon. Nine of these Bz alleles retained the transposon somewhere in their genome. In this study we show that like Ac (Schwartz 1989; Dooner and Belachew 1989), the 6.7 kb Ds element can transpose within a short physical distance, both proximal and distal to its original position. Additional bz sequences have been mapped immediately distal to the mutant locus in bz-m4 D6856; genetic evidence suggests these are flanked by two additional Ds elements. The remaining Bz revertant, Bz :107, arose from excision of a more complex 13 kb Ds element.     

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus

Summary A linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5 termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.