Resulting Hybrid Plasmids Research Articles

An Unorthodox Bacteriophytochrome from Rhodobacter sphaeroides Involved in Turnover of the Second Messenger c-di-GMP

Bacteriophytochromes are bacterial photoreceptors that sense red/far red light using the biliverdin chromophore. Most bacteriophytochromes work as photoactivated protein kinases. The Rhodobacter sphaeroides bacteriophytochrome BphG1 is unconventional in that it has GGDEF and EAL output domains, which are involved, respectively, in synthesis (diguanylate cyclase) and degradation (phosphodiesterase) of the bacterial second messenger c-di-GMP. The GGDEF-EAL proteins studied to date displayed either diguanylate cyclase or phosphodiesterase activity but not both. To elucidate the function of BphG1, the holoprotein was purified from an Escherichia coli overexpression system designed to produce biliverdin. The holoprotein contained covalently bound biliverdin and interconverted between the red (dark) and far red (light-activated) forms. BphG1 had c-di-GMP-specific phosphodiesterase activity. Unexpectedly for a photochromic protein, this activity was essentially light-independent. BphG1 expressed in E. coli was found to undergo partial cleavage into two species. The smaller species was identified as the EAL domain of BphG1. It possessed c-di-GMP phosphodiesterase activity. Surprisingly, the larger species lacking EAL possessed diguanylate cyclase activity, which was dependent on biliverdin and strongly activated by light. BphG1 therefore is the first phytochrome with a non-kinase photoactivated enzymatic activity. This shows that the photosensory modules of phytochromes can transmit light signals to various outputs. BphG1 is potentially the first "bifunctional" enzyme capable of both c-di-GMP synthesis and hydrolysis. A model for the regulation of the "opposite" activities of BphG1 is presented.

Loss of Outer Membrane Proteins without Inhibition of Lipid Export in an Escherichia coli YaeT Mutant

Escherichia coli yaeT encodes an essential, conserved outer membrane (OM) protein that is an ortholog of Neisseria meningitidis Omp85. Conflicting data with N. meningitidis indicate that Omp85 functions either in assembly of OM proteins or in export of OM lipids. The role of YaeT in E. coli was investigated with a new temperature-sensitive mutant harboring nine amino acid substitutions. The mutant stops growing after 60 min at 44 degrees C. After 30 min at 44 degrees C, incorporation of [35S]methionine into newly synthesized OM proteins is selectively inhibited. Synthesis and export of OM phospholipids and lipopolysaccharide are not impaired. OM protein levels are low, even at 30 degrees C, and the buoyant density of the OM is correspondingly lower. By Western blotting, we show that levels of the major OM protein OmpA are lower in the mutant in whole cells, membranes, and the growth medium. SecA functions as a multicopy suppressor of the temperature-sensitive phenotype and partially restores OM proteins. Our data are consistent with a critical role for YaeT in OM protein assembly in E. coli.

A novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in Escherichia coli.

A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of Clostridium acetobutylicum and the two PHA synthase genes (phaE and phaC) of Thiocapsa pfennigii in Escherichia coli. The four genes were cloned into the BamHI and EcoRI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to E. coli JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.

Expression and secretion of human lipocortin-1 by promoter and signal sequence of STA1 from Saccharomyces diastaticus

For the secretion of human lipocortin-1 (LC-1) in yeast, a expression and secretion vector was constructed by using the promoter and signal sequence of glucoamylase gene (STA1) of Saccharomyces diastaticus. After the cDNA of human LC-1 was ligated with the secretion vector, the resulting hybrid plasmid was transformed into S. diastaticus. When the recombinant S. diastaticus was cultivated in YPD medium, LC-1 was expressed and secreted into the extracellular medium, yielding LC-1 protein at a concentration of 2.5 μg/mL.

A new promoter‐probe vector for Saccharomyces cerevisiae using fungal glucoamylase cDNA as the reporter gene

A system is described for the selection of DNA sequences showing promoter activity in the yeast Saccharomyces cerevisiae using a heterologous reporter enzyme which is efficiently secreted by the yeast host. A multicopy shuttle plasmid of the YEp-type was constructed so as to carry multiple unique cloning sites at the 5' end of the Aspergillus awamori glucoamylase cDNA. Glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a DNA fragment from any source, provided it is endowed with promoter function in S. cerevisiae. As the glucoamylase signal-peptide is functional in S. cerevisiae, the enzyme is efficiently secreted by the yeast transformants. This phenotype can be very easily detected on plate assays and accurately quantified by spectrophotometric analysis of the culture supernatant. Since S. cerevisiae naturally lacks amylolytic activity, any wild-type strain can be used as a host in this system. To evaluate the system, a DNA pool of random fusions was created by ligating sau 3A digested S. cerevisiae genomic DNA to the BglII-linearized vector. The resulting hybrid plasmids were transformed into S. cerevisiae and several transformants secreting glucoamylase to varying degrees were obtained.

Identification of the DNA region responsible for sulfur-oxidizing ability of Thiosphaera pantotropha

For the identification of the DNA region responsible for the sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha, we used previously isolated Tn5-mob insertional Sox- mutants. For seven mutants, the Tn5-mob insertion was localized on the chromosome rather than on the megaplasmids pHG41 or pHG42 by using the Tn5-mob-harboring vehicle pSUP5011 as probe. The specific insertion of Tn5-mob into a sox gene was determined for one Sox- mutant, strain TP19. An 18-kb EcoRI fragment was cloned in Escherichia coli by using the mobilizable plasmid pSUP202 as vector and the kanamycin resistance gene of Tn5 as marker. Conjugal transfer of the resulting hybrid plasmid, pKS3-13, to the wild type resulted in two phenotypically different groups of recombinants. Ninety-five percent of the recombinants were Sox+, kanamycin resistant, and tetracycline resistant; 5% were homogenote recombinants exhibiting the Sox-, kanamycin-resistant, tetracycline-sensitive phenotype, and these indicated the specific insertion. To isolate the respective wild-type sox gene, total DNA from a heterogenote recombinant was partially restricted with EcoRI, religated, and transformed in E. coli. Transformants carrying a pSUP202-derived hybrid plasmid with the intact sox gene were identified by screening for a tetracycline-resistant, kanamycin-sensitive, and chloramphenicol-sensitive phenotype and by complementation of the Sox- mutant TP19. A plasmid of this type, pEG12, contained an insert of 13 kb which gave a positive signal in Southern hybridization with the homologous probe of pKS3-13. pEG12 was used to determine the DNA homology of the sulfur-oxidizing enzyme systems of other thiobacteria. Strong hybridization signals were obtained with total DNA of the neutrophilic sulfur-oxidizing bacteria Paracoccus denitrificans, Thiobacillus versutus, and Rhodobacter capsulatus. No hybridization signal was obtained with DNA of other neutrophilic or acidophilic thiobacteria examined.

Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene

Synthetic oligodeoxynucleotide probes based on the known amino acid sequence of Rhodobacter sphaeroides Y thioredoxin were used to identify, clone, and sequence the structural gene. The amino acid sequence derived from the DNA sequence of the R. sphaeroides gene was identical to the known amino acid sequence of R. sphaeroides thioredoxin. An NcoI site was created by directed mutagenesis at the beginning of the thioredoxin gene, inducing in the encoded protein the replacement of serine in position 2 by alanine. The 421-base-pair NcoI-PstI restriction fragment obtained was ligated in the pKK233-2 expression vector and the resulting hybrid plasmid was used to transform Escherichia coli strains lacking functional thioredoxin. Transformants that complemented mutations in the trxA gene were identified by increased colony size on rich medium, growth on minimal medium with methionine sulfoxide, and ability to support M13 growth and T7 replication; this latter phenotype implies correct interaction between R. sphaeroides thioredoxin and the product of T7 gene 5. The presence of R. sphaeroides thioredoxin was further confirmed by enzyme assay.

Isolation of fragments with pac function for phage P22 from phage LP7 DNA and comparison of packaging gene 3 sequences

Three PstI DNA fragments of the P22-related Salmonella phage, LP7, have been cloned. They contain sequences recognized as pac signals by the packaging apparatus of P22. One of these fragments corresponds to the P22 DNA fragment carrying gene 3 which comprises the pac signal of phage P22. The product of gene 3, Gp3, is involved in the recognition of pac and the packaging process. Gene 3 of LP7 and most of the adjacent gene 2 have been sequenced. The pac analogous segments of the other two PstI fragments have been narrowed down by subcloning and by transduction of the resulting hybrid plasmids under recombination-defective conditions.

Codon usage as a reason for unsuccessful search for amber-suppressor mutants inStreptomyces lividans?

SummaryAn amber mutation was created in the CAT gene of plasmid vector pACYC184 and this modified plasmid was fused with theStreptomycesvector pIJ702 for use as an indicator for the identification ofStreptomycesstrains carrying nonsense suppressor tRNA mutations. The resulting hybrid plasmid pGM1109 was introduced into the chloramphenicol-sensitive mutant M252 ofStreptomyces lividans. Chloramphenicol-resistant colonies were isolated and characterized. None of them was a nonsense suppressor mutant. The failure to obtain such mutants is discussed on the basis of codon usage in streptomycetes.

The plasmids of the morels: characterization and prerequisites for vector development

In several Morchella species linear dsDNA plasmids have been discovered and analyzed. Two linear plasmids of the strain Morchella conica 3 analyzed in detail are associated with the mitochondria but not as an integrative part of the high molecular weight mt DNA. In addition, homologies between different plasmids from different species could be detected. These homologies extend to all parts of the plasmids. Differences in size are due to different lengths of nonhomologous inner parts rather than to differences at the termini. Fragments comprising the entire plasmids were cloned into bacterial vectors and a selectable marker gene for eukaryotes was added to the resulting hybrid plasmids. Transformation of yeast led to the identification of autonomously replicating sequences, this being a prerequisite for the development of autonomously replicating vectors for Morchella.

Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

The gene ( prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting in a DNA fragment of approx. 1.8kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an M r of approx. 40 000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up to nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA.

Glutathione production by Escherichia coli cells with hybrid plasmid containing tandemly polymerized genes for glutathione synthetase

A DNA fragment containing the structural and promoter regions of glutathione synthetase (GSH II) gene (gsh II) from Escherichia coli B were polymerized. The dimeric and trimeric DNA fragments obtained were inserted into Bam HI site of vector plasmid pBR325 and the resulting hybrid plasmids were designated pGS401-02 and pGS401-03, respectively. The GSH II activity of E. coli cells with these hybrid plasmids increased depending on the number of the genes (gsh II) contained. To construct hybrid plasmids useful for glutathione production, another DNA fragment with a gene (gsh I) for γ-glutamylcysteine synthetase (GSH I) from E. coli B was inserted into Pst I sites of pGS401-02 and pGS401-03 and the hybrid plasmids obtained (pGS501-12 and pGS501-13, respectively) were introduced into E. coli B cells. Although the glutathione-producing activities of the cells with these plasmids were little improved as compared with that of cells with the hybrid plasmid (pGS501-11) containing both gsh I and gsh II because of the low activity of GSH I, our method has brought to light a new type of gene amplification.

Characterization of a cryptic plasmid (p0M1) inButyrivibrio fibrisolvens by restriction endonuclease analysis and its cloning inEscherichia coli

A small cryptic plasmid has been identified in a strain of the ruminal bacteriumButyrivibrio fibrisolvens. This plasmid has been isolated and purified. It is approximately 2.8 kbp in length and contains restriction sites for a number of common endonucleases including single sites for EcoRI, PvuII, and PstI. A map of the plasmid restriction sites has been constructed. This plasmid, designated p0M1, has been ligated to pBR325, pAT153, and pHV33 and transformed intoEscherichia coli, and the resulting hybrid plasmids have been mapped. The possible uses of such hybrid plasmids for gene cloning inB. fibrisolvens are discussed.

Application of a plasmid carrying a gene for orotate phosphoribosyltransferase (pyrE) to orotidine-5'-monophosphate production in Escherichia coli K-12.

Two kilobase (2.0 kb) DNA fragments bearing a gene (pyrE) for orotate phosphoribosyltransferase (OPTase) were cloned into pBR322, from both Escherichia coli K-12 C600 and its derivative, purine-sensitive mutant PS 100 (caused by a pyrE mutation). Introduction of the resulting hybrid plasmids (designated as pNE24 with DNA from C600, and pNE31 with DNA from PS 100) into E. coli K-12 increased the amount of OPTase in the host cells to 30~80 fold. The PS 100 cells with pNE31 overproduced a mutant-type OPTase with a decreased level of activity as compared to the wild-type OPTase. The purine-sensitive phenotype of PS 100 was completely suppressed by introduction of either pNE24 or pNE31. In a reaction involving the conversion of orotate to orotidine-5′-monophosphate (OMP) by an E. coli crude extract, the increased OPTase activity on the use of the hybrid plasmid, pNE24, was favorable for a high conversion rate.

Definition of oriR, the minimum DNA segment essential for initiation of R1 plasmid replication in vitro.

The 3.6-kilobase Bgl II-EcoRI fragment from R1 plasmid containing copA, repA, and the replication origin (ori) was inserted into the ColE1-type plasmid pUC8. The resulting hybrid plasmid replicates in extracts prepared from both polA- and polA+ cells, whereas pUC8 replicates only in a polA+ extract. This characteristic provides a method for assaying the repA and ori functions. Hybrid plasmids that were either repA- or ori- were unable to replicate in a polA- cell extract. Replication of the repA- ori+ plasmid was restored by complementation of the repA defect by a repA+ ori- plasmid in vitro. Successful complementation of the repA function in vitro provides a method for assaying the repA protein. In order to define the minimum DNA segment with origin function (oriR), deletions were introduced starting from either side of the insert, and the replication properties of the plasmids carrying these deletions were examined in a polA- cell extract. The right end of oriR was located at position 1,611 in the nucleotide coordinates defined previously [Ryder, T., Rosen, J., Armstrong, K., Davidson, D. & Ohtsubo, E. (1981) in The Initiation of DNA Replication: ICN-UCLA Symposia on Molecular and Cellular Biology, ed. Ray, D.S. (Academic, New York), Vol. 22, 91-111]. By complementing repA- ori+ plasmids with the repA+ ori- plasmid, the left end of oriR was localized at position 1,424. Therefore, the oriR sequence, localized within a region of 188 base pairs, is separate from the repA gene. A hybrid plasmid carrying the 206-base-pair segment between positions 1,406 and 1,611 also replicates in a polA- cell extract when the repA function is supplied in trans. Removal of an additional 66 base pairs (positions 1,406-1,471) inactivates the function of the minimal oriR segment.

Chromosomal locations of the genes for rRNA in Escherichia coli K-12.

Chromosomal locations of the seven rRNA operons in Escherichia coli K-12 were studied by digesting DNA from various merodiploid strains with SalI restriction enzyme followed by Southern gel analysis with 32P-labeled 23S rRNA as a probe. The seven unique SalI DNA fragments revealed in the autoradiograms were first correlated to the seven rRNA operons previously isolated as hybrid plasmids or transducing phages. The chromosomal locations of six (rrnA, B, C, D, E, and G) of the seven isolated operons were confirmed by increased gene dosage demonstrated in autoradiograms after Southern gel analysis of DNA from relevant merodiploid strains. The gene dosage analysis showed that the location of the remaining operon (now called rrnH) is between metD and proA. No evidence was obtained for the presence of rrnF, which was previously reported to map between aroB and malA. The chromosomal location of rrnH was confirmed by P1 transduction in the following way: a DNA fragment adjacent to rrnH was cloned into pBR322; the resulting hybrid plasmid was integrated at the homologous region of the chromosome of a polA mutant; and the ampicillin resistance marker originally carried by pBR322 was then used for mapping of the nearby rrnH by P1 transduction. A close linkage of rrnH to metD (about 60% cotransduction) was observed, and the data were consistent with the order metD-rrnH-proA. Thus, mapping of all seven rRNA operons has been completed. The present study has also determined the orientation of rrnG and rrnH and demonstrated that the direction of transcription of all the rRNA operons is identical to that of DNA replication.

Cloning of genes involved in membrane lipid synthesis: effects of amplification of phosphatidylglycerophosphate synthase in Escherichia coli.

The structural gene (pgsA) for the CDP-diacylglycerol:sn-glycero-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycerophosphate synthase) from Escherichia coli has been cloned, using pSC101 as the vector. The resulting hybrid plasmids not only correct the lack of in vitro synthase activity in pgsA strains but also cause an amplification (6- to 40-fold over wild-type levels) in enzymatic activity in direct proportion to the copy number of the plasmids found in vivo. The cloned gene also corrects the abnormally low level of polyglycerophosphatides found in pgsA strains and actually increases the level of phosphatidylglycerol to above that normally found in E. coli. The degree of alteration in phospholipid composition brought about by these hybrid plasmids is not of the order expected if fluctuations in enzyme levels in vivo were an important regulatory mechanism in phospholipid metabolism. The isolated hybrid plasmids have been mapped by restriction endonuclease analysis. The presence and location of other genetic markers have also been established. The above data, along with analysis of deletion derivatives of these plasmids and subcloning of appropriate restriction fragments, have established the position of the pgsA locus on the hybrid plasmids. From this data, the position of the pgsA locus has been determined to le between flaI and uvrC on the E. coli genetic map.

Naturally occurring macrolide-lincosamide-streptogramin B resistance in Bacillus licheniformis.

Resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics is widespread and of clinical importance. B. Weisblum and his coworkers have demonstrated that this resistance is associated with methylation of the 23S ribosomal ribonucleic acid of the large ribosomal subunit which results in a diminished affinity of this organelle for these antibiotics (Lai et al, J. Mol. Biol. 74:67-72, 1973). We report that 10 of 15 natural isolates of Bacillus licheniformis, a common soil organism, are resistant to the MLS antibiotics. The properties of this resistance (high level of tolerance for erythromycin, broad cross-resistance spectrum, and inducibility) suggest that resistance is conferred as described above. The resistance determinant from one of these strains was cloned onto a B. subtilis plasmid vector, and the resulting hybrid plasmid (pBD90) was used to prepare radioactive probe deoxyribonucleic acid for hybridization studies. All of the resistance B. licheniformis strains studied exhibited homology with the pBD90 insert. Plasmid pBD90 showed no homology to the following staphylococcal and streptococcal MLS-resistance plasmids: pE194, pE5, pAM77, pI258. Plasmids pE194 and pE5, on the other hand, carry homologous MLS genes but showed no detectable homology to one another in their replication genes. pBD90 specified a 35,000-dalton erythromycin-inducible protein, detectable in minicells, which therefore appears different from the 29,000-dalton inducible resistance protein specified by pE194. We conclude that there are at least three distinct MLS resistance determinants to be found among gram-positive bacteria.

Inversions of specific DNA segments in flagellar phase variation of Salmonella and inversion systems of bacteriophages P1 and Mu.

Prophages P1 and Mu produces a trans-acting factor possessing the din+ activity which catalyzes the inversion of the specific DNA segment responsible for flagellar phase variation of Salmonella, din mutants were isolated from PICMclr100 phage by selecting phages that did not suppress the yh2 mutation of Salmonella in prophage state. No inversion loop structure was detected among DNA forms arising after denaturation and rehybridization of DNAs extracted from the din mutants. The DNA fragment containing C region of P1 was cloned on a plasmid vector, pCR1. The resulting hybrid plasmid, pKK2, was shown to possess the din+ activity: the vh2 mutant of Salmonella harboring the plasmid changed the flagellar phase. From analysis of the plasmid by use of BamHI and Bgl II, the din gene specifying the din+ activity was located near or within the C region of P1. It is highly plausible that the din gene of P1 was also involved in the inversion of the C region. Similarly, the DNA fragment containing the G and beta segments of Mu was cloned on pCR1. The resulting hybrid plasmid, pII101, also possessed the din+ activity.

Molecular cloning, correlation of genetic and restriction maps, and determination of the direction of transcription of gnd of Escherichia coli.

Expression of the gene gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, is regulated by growth rate. Using deoxyribonucleic acid from the specialized transducing phage lambda h80 dgnd his as the source of gnd, we cloned restriction fragments carrying the complete gene and portions of it on the plasmid vector pBR322. A hybrid plasmid carrying a 3.7-megadalton HindIII restriction fragment from the phage was prepared and found to be gnd+. Through restriction mapping of this fragment and subcloning segments of it, we prepared a gnd+ hybrid plasmid which carried only 1.85 megadaltons of E. coli deoxyribonucleic acid. A cleavage site for the restriction endonuclease PstI was located on the genetic map of gnd by cloning adjacent EcoRI-PstI restriction fragments and crossing the resulting hybrid plasmids with previously mapped gnd deletion and bacteriophage Mu insertion mutants. A maxicell experiment was used to determine the direction of transcription of gnd, to identify which EcoRI-PstI fragment contains the gnd promote, and to localize th beginning of the structural gene to a region about 850 +/- 150 base pairs from the PstI cleavage site. A fine-structure restriction map surrounding the PstI cleavage site was prepared for endonucleases KpnI, HincII, HaeIII, HpaII, and TaqI.