Growth-phase-dependent induction of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase in the cyanobacterium Synechococcus sp. PCC7942.

In most cyanobacteria, the only known pathway for oxidation of stored carbohydrate in the dark or under energy-limiting conditions is the hexose monophosphate shunt. To determine whether the increased use of the shunt under these conditions derives from an increase in the activity level of the respective enzymes, we measured the effect of growth phase during the growth of batch cultures of Synechococcus sp. strain PCC7942 on the specific activity of 6-phosphogluconate dehydrogenase (6PGD) and glucose 6-phosphate dehydrogenase. The specific activities were constant during the exponential growth phase of the culture, but they increased about fivefold during the transition into stationary phase. As an approach to determining the level of expression at which the growth-phase-dependent regulation of 6PGD level is exerted, we constructed operon and gene fusions between the gnd gene, which encodes 6PGD, and the Escherichia coli lacZ gene, which encodes beta-galactosidase (beta Gal). Strains harboring the fusions integrated into the cyanobacterial chromosome were prepared, and the growth-phase dependence of beta Gal level was determined. The specific activity of beta Gal in cultures of both types of fusion strains increased during the transition into stationary phase, indicating that the growth-phase-dependent regulation is on the gnd mRNA level. Characterization of the growth-phase-dependent induction of 6PGD in strains carrying differing amounts of DNA upstream from the gnd structural gene led to the localization of the promoter and the regulatory site on the restriction map of the gene, whose sequence has previously been determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth-rate-dependent expression and cloning of gnd alleles from natural isolates of Escherichia coli.

6-Phosphogluconate dehydrogenase (6PGD), encoded by gnd, is highly polymorphic among isolates of Escherichia coli form natural populations. As a means of characterizing the growth-rate-dependent regulation of the level of 6PGD, five gnd alleles, including the E. coli B/r allele, were crossed into E. coli K-12 with bacteriophage P1. In each of the isogenic strains, the level of 6PGD was two- to threefold higher in cells grown on glucose than in cells grown on acetate. The level of enzyme activity in the acetate-grown cells varied about sixfold within the set of isogenic strains. The physiological importance of these differences in enzyme level is discussed. The gnd gene was cloned from five E. coli strains and Salmonella typhimurium LT-2 and mapped with twelve restriction endonucleases. gnd was located and oriented on the chromosomal DNAs. The restriction maps of the genes were aligned at conserved restriction sites, and the relative divergence of the genes was estimated from restriction site polymorphisms. The E. coli gnd genes differed from the S. typhimurium gene by about 11%. Most of the E. coli genes differed from one another by less than 5%, but one allele differed from the others by about 10%. Only the gnd gene from E. coli K-12 had an IS5 element located nearby.     

Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties.

Biphasic, chimeric plasmids for the transformation of Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain 7002) were constructed by splicing the 3.0-megadalton cryptic plasmid from strain PR-6 into plasmids pBR322 and pBR325 from Escherichia coli. Transformants of either E. coli or strain PR-6 by these plasmids could be detected on the basis of the drug resistance marker(s) carried by the chimeric plasmids. Plasmid DNA isolated from a PR-6 transformant transformed PR-6 much more efficiently than plasmid DNA prepared from E. coli. Plasmids from which the AvaI recognition site was deleted (AvaI is an isoschizomer of the AquI restriction endonuclease of strain PR-6) also transformed strain PR-6 much more efficiently than did plasmids containing the AvaI recognition site. These and other results suggest that AquI strongly effects plasmid transformation when the donor plasmid contains an unmodified AquI recognition site. Multimeric forms of the chimeric plasmids are also much more efficient at transforming strain PR-6 than are the analogous monomeric forms.     

Specific Deletion Occurring in the Directed Evolution of 6-Phosphogluconate Dehydrogenase in ESCHERICHIA COLI

A novel genetic change leading to increased activity of 6-phosphogluconate dehydrogenase (6PGD) in E. coli has been observed. The mutation is a deletion of approximately 0.4 kilobase pairs occurring between the structural gene of 6PGD (gnd) and one copy of an insertion element (IS5) found normally in E. coli K12 a few hundred base pairs upstream (counterclockwise) from gnd at 44 minutes on the conventional genetic map. The deletion is associated with a threefold higher activity of 6PGD and a 57% increase in the maximum growth rate when cells are grown in gluconate.     

Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant.

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.     

Cloning and expression of the thermostable pullulanase gene from Thermus sp. strain AMD-33 in Escherichia coli

Abstract The gene coding for a thermostable pullulanase from a thermophile, Thermus sp. strain AMD-33, was cloned in Escherichia coli using pDR540 as a vector. A restriction map was determined for the plasmid pTPS131 which contained the fragment carrying the pullulanase gene. DNA-DNA hybridisation analysis showed that the DNA fragment contained the gene from Thermus sp. strain AMD-33. The strain of E. coli harbouring the plasmid pTPS131 produced most of the pullulanase protein cellularly, whereas Thermus sp. strain AMD-33 produced pullulanase extracellularly. Comparative studies of the enzyme from the thermophile and the plasmid-encoded enzyme in E. coli demonstrated that the optimum temperature and pH of the enzymes were closely similar.     

Cloning the KpnI restriction-modification system in Escherichia coli

The genes encoding the KpnI restriction and modification (R-M) system from Klebsiella pneumoniae, recognizing the sequence, 5'-GGTAC decreases C-3', were cloned and expressed in Escherichia coli. Although the restriction endonuclease (ENase)- and methyltransferase (MTase)-encoding genes were closely linked, initial attempts to clone both genes as a single DNA fragment in a plasmid vector resulted in deletions spanning all or part of the gene coding for the ENase. Initial protection of the E. coli host with MTase expressed on a plasmid was required to stabilize a compatible plasmid carrying both the ENase- and the MTase-encoding genes on a single DNA fragment. However, once established, the MTase activity can be supplied in cis to the kpnIR gene, without an extra copy of kpnIM. A chromosomal map was generated localizing the kpnIR and kpnIM genes on 1.7-kb and 3.5-kb fragments, respectively. A final E. coli strain was constructed, AH29, which contained two compatible plasmids: an inducible plasmid carrying the kpnIR gene which amplifies copy number at elevated temperatures and a pBR322 derivative expressing M.KpnI. This strain produces approx. 10 million units of R.KpnI/g of wet-weight cells, which is several 1000-fold higher than the level of R.KpnI produced by K. pneumoniae. In addition, DNA methylated with M.KpnI in vivo does not appear to be restricted by the mcrA, mcrB or mrr systems of E. coli.     

Intramolecular transposition and inversion in plasmid R6K

Selection was made in Escherichia coli K-12 recA hosts carrying plasmid R6K for ampicillin hyperresistance. Twenty-two selected strains were found to carry mutant plasmids, which, from electron microscopy and restriction enzyme analysis, were concluded to arise by a duplication of transposon Tn2660, which confers ampicillin resistance, in all cases the duplicate transposon being in an inverted orientation with respect to the resident Tn2660. A mutant of R6K, pSJC301, which was temperature sensitive for ampicillin resistance was produced by in vitro hydroxylamine treatment of R6K deoxyribonucleic acid. A plasmid hybrid, pSJC102, was constructed by cloning the EcoRI R6K fragment carrying the wild-type beta-lactamase gene into the EcoRI site of ColE1. pSJC301 and pSJC102 were transformed into the same recA host strain to form a stable biplasmid strain. Ampicillin-hyperresistant mutants were selected from this strain and screened for plasmids with a duplication of transposon Tn2660, which occurred with equal frequency in either pSJC301 or pSJC102; of 12 characterized, all were inverse repeats of the resident transposon. All six Tn2660 inserts into pSJC301 determined temperature-sensitive ampicillin resistance, and all six inserts into pSJC102 determined wild-type ampicillin resistance, from which it was inferred that transposition of a duplicate Tn2660 occurs predominantly as an intramolecular event, at least in the multicopy R6K plasmid. In all 28 insertion mutants of R6K, there was an inversion of the deoxyribonucleic acid between the two transposons, whereas in only one of six insertion mutants of pSJC102, inversion had occurred. These results are discussed in terms of current models of transposition.     

Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204.

A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.     

Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2

The blue-green alga, Synechococcus R2, is transformed to antibiotic resistance by chimeric DNA molecules consisting of Synechococcus R2 chromosomal DNA linked to antibiotic-resistance genes from Escherichia coli. Chimeric DNA integrates into the Synechococcus R2 chromosome by homologous recombination. The efficiency of transformation, as well as the stability of integrated foreign DNA, depends on the position of the foreign genes relative to Synechococcus R2 DNA in the chimeric molecule. When the Synechococcus R2 DNA fragment is interrupted by foreign DNA, integration occurs through replacement of chromosomal DNA by homologous chimeric DNA containing the foreign insert; transformation is efficient and the foreign gene is stable. Mutagenesis in some cases attends integration, depending on the site of insertion. Foreign DNA linked to the ends of Synechococcus R2 DNA in a circular molecule, however, integrates less efficiently. Integration results in duplicate copies of Synechococcus R2 DNA flanking the foreign gene and the foreign DNA is unstable. Transformation in Synechococcus R2 can be exploited to modify precisely and extensively the genome of this photosynthetic microorganism.     

Stepwise cloning and genetic organization of the seemingly unclonable HgiCII restriction-modification system from Herpetosiphon giganteus strain Hpg9, using PCR technique.

The genes, hgiCIIR and hgiCIIM, that encode the HgiCII restriction and modification (R-M) system from Herpetosiphon giganteus strain Hpg9, an AvaII isoschizomer recognizing the sequence, GGATCC, were cloned in Escherichia coli. Cloning the respective hgiCIIM gene was achieved via in vitro selection both from a Sau3AI- and an NheI-generated plasmid gene library using AvaII, a commercially available isoschizomer of HgiCII. However, all attempts to clone the closely linked hgiCIIR and M genes in a single step resulted in deletions spanning parts of the coding region of hgiCIIR. Therefore, cloning of the missing 3'-terminal part of this gene was achieved by applying the inverse polymerase-chain-reaction technique. All attempts to construct an enzymatically active R.HgiCII failed; only the inactivated hgiCIIR gene could be cloned. Sequencing of the hgiCIIRM region (carrying predesigned small mutations in the R gene) disclosed three open reading frames (ORFs): one small ORF preceding the methltransferase (MTase)-encoding gene, plus those encoding M.HgiCII (49,620 Da) and R.HgiCII (30,891 Da). M.HgiCII exhibits the common motif of ten conserved amino-acid blocks typically found within the group of m5C-MTases. The R-M system of HgiCII reveals strong homologies to the isoschizomeric R-M system of HgiBI from H. giganteus strain Hpg5, which, in contrast, could be cloned in one step.     

Chloramphenicol acetyltransferase expression in marine Rhodobacter sp. NKPB 0021 by use of shuttle vectors containing the minimal replicon of an endogenous plasmid

A vector, pUK318, was constructed to allow the expression of foreign genes in the marine photosynthetic bacterium Rhodobacter sp. NKPB 0021. This strain has been cured of its two endogenous plasmids. pUK318 consists of a 2.3-kb PstI-BamHI restriction fragment, containing a marine Rhodobacter plasmid replication region, cloned into pUC18. This fragment was derived from plasmid pRD31, a 3.1-kb endogenous plasmid purified from the marine strain Rhodobacter sp. NKPB 043402. A kanamycin resistance gene from Tn903 was cloned into the PstI restriction site to provide antibiotic selection. pUK318 was transferred to Rhodobacter sp. NKPB 0021 by transformation, and efficiencies of 7.2 x 10(-5) were obtained. Furthermore, pUK318 was stably maintained when transformants were grown either heterotrophically or photosynthetically in the absence of antibiotics. pUK318 was used to express the Escherichia coli chloramphenicol acetyl transferase (CAT) gene in Rb. NKPB 0021. Transformants expressed a maximum CAT activity of 1.12 mmol/min/g dry cells. In addition, the DNA region essential for pUK318 replication in Rb. NKPB 0021 was localized to a 1.36-kb HincII-PstI fragment. This is the first report of a plasmid vector containing a marine Rhodobacter-specific replicon that allows stable expression of foreign genes in the absence of antibiotic selection.     

Identification,cloning, and sequencing of the genes involved in the conversion of D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp. strain ON-4a

The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.     

Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli encodes O6-methylguanine-DNA methyltransferase, which serves as a positive regulator of the adaptive response to alkylating agents and as a DNA repair enzyme. The gene which can make an ada-deficient strain of E. coli resistant to the cell-killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the gene potentially encoded a protein with a calculated molecular weight of 39,217. Since the nucleotide sequence of the cloned gene shows 70% similarity to the ada gene of E. coli and there is an ada box-like sequence (5'-GAATTAAAACGCA-3') in the promoter region, we tentatively refer to this cloned DNA as the adaST gene. The gene encodes Cys-68 and Cys-320, which are potential acceptor sites for the methyl group from the damaged DNA. The multicopy plasmid carrying the adaST gene significantly reduced the frequency of mutation induced by MNNG both in E. coli and in S. typhimurium. The AdaST protein encoded by the plasmid increased expression of the ada'-lacZ chromosome fusion about 5-fold when an E. coli strain carrying both the fusion operon and the plasmid was exposed to a low concentration of MNNG, whereas the E. coli Ada protein encoded by a low-copy-number plasmid increased it about 40-fold under the same conditions. The low ability of AdaST to function as a positive regulator could account for the apparent lack of an adaptive response to alkylation damage in S. typhimurium.     

Broad-host-range IncP-4 plasmid R1162: effects of deletions and insertions on plasmid maintenance and host range

R1162 is an 8.7-kilobase (kb) broad-host-range replicon encoding resistance to streptomycin and sulfa drugs. In vitro deletion of 1.8-kb DNA between coordinates 3.0 and 5.3 kb did not affect plasmid maintenance, but a Tn1 insertion at coordinate 6.3 kb led to a recessive defect in plasmid maintenance. The only cis-acting region necessary for plasmid replication appears to lie between the Tn1 insertion at coordinate 6.3 kb and a second Tn1 insertion at coordinate 6.5 kb. All R1162 sequences between position 6.5 kb and the EcoRI site at coordinate 8.7/0 kb were dispensible for replication in Escherichia coli and Pseudomonas putida. Plasmids carrying insertions in a variety of restriction sites in an R1162::Tn1 derivative were unstable in P. putida but stable in E. coli. Tn5 insertions in R1162 showed a hot spot at coordinate 7.5 kb. A Tn5 insertion at coordinate 8.2 kb appeared to mark the 3' end of the streptomycin phosphotransferase coding sequence. All R1162::Tn5 derivatives showed specific instability in Pseudomonas strains but not in E. coli. The instability could be relieved by internal deletions of Tn5 sequences. In the haloaromatic-degrading Pseudomonas sp. strain B13, introduction of an unstable R1162::Tn5 plasmid led to loss of ability to utilize m-chlorobenzoate as a growth substrate. Our results showed that alteration of plasmid sequence organization in nonessential regions can result in restriction of plasmid host range.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.     

Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O antigen of an E. coli O2: K1 strain

Abstract: The rfb gene cluster which determines the biosynthesis of the O2 O antigen has been cloned from an Escherichia coli O2: K1 strain isolated from a case of septicaemia in chickens. The region required for expression of the O antigen in E. coli K-12 was localised to a 10.7 to 14.15-kb segment which was shown to be chromosomal in origin with a close linkage to the gnd and his genetic loci.     

Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli

The gene coding for the sequence-specific modification methylase methM . BspI of Bacillus sphaericus R has been cloned in Escherichia coli by means of plasmid pBR322. The selection was based on the expression of the cloned gene which rendered the recombinant plasmid resistant to BspI restriction endonuclease cleavage. The gene is carried by a 9 kb BamHI fragment and by a smaller 2.5 kb EcoRI fragment derived from the BamHI fragment. The Bsp-specific methylase level was found to be higher in the recombinant clones than in the parental strain. The methylase gene is probably located on the Bacillus sphaericus chromosome, and not on a plasmid known to be carried by this strain. The recombinant clones do not exhibit an BspI restriction endonuclease activity.