Cloning and characterization of a lysine decarboxylase gene from Hafnia alvei

Summary A lysine decarboxylase (LDC) gene from Hafnia alvei was cloned in the Escherichia coli strain HB101. A gene bank consisting of 2,000 clones, carrying recombinant plasmids with large DNA fragments of H. alvei integrated in the BamH1 site of pBR322, was screened for LDC activity by a colony filter radioimmunoassay. The gene bank yielded clone 462 expressing high LDC activity with the presence of a plasmid carrying a 7.5 kb insert of H. alvei. Two LDC-positive subclones derived from 462 with inserts of 2.9 and 3.3 kb were sequenced by the shotgun method. An open reading frame for a 83 K protein with 739 amino acids was determined as the coding region for the LDC. The identification of this reading frame as the true reading frame of the H. alvei LDC gene and its similarities with LDC of E. coli are described. The use of the cloned gene for the transformation of plant cells is discussed.     

Cloning and characterization of Rv0621 gene related to surfactant stress tolerance in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

To understand how Mycobacterium tuberculosis (M. tuberculosis) could survive in human lung, Genomic expression library of M. tuberculosis in Escherichia coli (E. coli) had been prepared. Taking advantage of the genetic simplicity of E. coli and the functional conservation of some prokaryote proteins, a surfactant stress resistant gene Rv0621 was identified, which encodes a 37 kDa putative membrane protein. The E. coli colony with the partial Rv0621 gene insert, named S1, was able to grow in medium containing 0.4% sodium dodecyl sulfate, while the strain carried empty vector was unable to grow. The full length of the Rv0621 gene was then cloned into plasmid pET32a (+) expressed in E. coli BL21 (DE3). Using gas chromatographic–mass spectrometric (GC–MS), the fatty acid composition of the E. coli BL21 (DE3) carrying Rv0621–pET32a (+) and the E. coli BL21 (DE3) carrying empty vector pET32a (+) were compared. E. coli BL21 (DE3) carrying Rv0621–pET32a (+) contained more oleic acid. This suggests the gene may be involved in regulation of fatty acid synthesis and M. tuberculosis resistance to the surfactant defense of its host.     

Cloning and high expression of glutaryl 7-aminocephalosporanic acid acylase gene from Pseudomonas diminuta

The gene coding for the glutaryl 7-aminocephalosporanic acid (GL 7-ACA) acylase from Pseudomonas diminuta KAC-1 was cloned and expressed in Escherichia coli. The acylase gene was composed of 2160 base pairs and encoded a polypeptide of 720 amino acid residues. The E. coli BL21 carrying pET2, the plasmid construct for high expression of GL 7-ACA acylase gene, produced this enzyme at approx. 30% of the total proteins with 3.2 units activity mg protein^–1. Growth at temperature below 31 °C and deletion of signal peptide increased the processing of precursor acylase to active enzyme in the recombinant E. coli cells.     

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

Summary A recombinant phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec^– phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.     

Purification and Some Properties of Urethane Hydrolase II from Lactobacillus casei ω ATCC 7469

The β-1,3-glucanase (1,3-β-d-glucan glucanohydrolase, EC 3.2.1.6) gene from Flavobacterium dormitator var. glucanolyticae was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKUβG1 (8.2 kb), showed a high β-1,3-glucanase activity and a lytic activity on viable yeast cells. These activities were found in the peripiasmic space of E. coli clone cells. Southern hybridization analysis showed that the cloned gene was derived from F. dormitator chromosomal DNA. The gene products were purified from the periplasmic fraction of E. coli by ammonium sulfate fractionation and ion-exchange chromatography. The purified enzymes were demonstrated to be identical with a lytic endo-β-1,3-glucanase II and a nonlytic endo-β-1,3-glucanase I from F. dormitator from their enzymological and immunological properties. In the E. coli cells, endo-β-1,3-glucanase I was also formed by a proteolytic digestion of endo-β-1,3-glucanase II during the cultivation as in F. dormitator. Thus, the only endo-β-1,3-glucanase II was coded for in the cloned gene.     

Functional analysis of combinations in astaxanthin biosynthesis genes from<Emphasis Type="Italic">Paracoccus haeundaensis</Emphasis>

Carotenoids are important natural pigments produced by many microorganisms and plants. We have previously reported the isolation of a new marine bacterium,Paracoccus haeundaensis, which produces carotenoids, mainly in the form of astaxanthin. The astaxanthin biosynthesis gene cluster, consisting of six carotenogenic genes, was cloned and characterized from this organism. Individual genes of the carotenoid biosynthesis gene cluster were functionally expressed inEscherichia coli and each gene product was purified to homogeneity. Their molecular characteristics, including enzymatic activities, were previously reported. Here, we report cloning the genes for crtE, crtEB, crtEBI, crtEBIY, crtEBIYZ, and crtEBI-YZW of theP. haeundaensis carotenoid biosynthesis genes inE. coli and verifying the production of the corresponding pathway intermediates. The carotenoids that accumulated in the transformed cells carrying these gene combinations were analyzed by chromatographic and spectroscopic methods.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.     

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli

Summary The NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.Abbreviations GDH glutamate dehydrogenase - PMS phenazine methosulphate - MTT 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium-bromide - PMSF phenylmethylsulphonylfluoride - SSC standard saline citrate - DTT dithiothreitol - bp base pairs - kbp kilo base pairs - dNTP deoxynucleoside triphosphate     

Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis

The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. the gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239 bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).     

Two streptokinase genes are expressed with different solubility in Escherichia coli W3110

The streptokinase (SK) gene from S. equisimilis H46A (ATCC 12449) was cloned in E. coli W3110 under the control of the tryptophan promoter. The recombinant SK, which represented 15% of total cell protein content, was found in the soluble fraction of disrupted cells. The solubility of this SK notably differed from that of the product of the SK gene from S. equisimilis (ATCC 9542) which had been cloned in E. coli W3110 by using similar expression vector and cell growth conditions, and occurred in the form of inclusion bodies.     

Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli

A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.     

Genetic cloning and functional expression in Escherichia coli of an archaerhodopsin gene from Halorubrum xinjiangense

Pairs of PCR primers that targeted the archae/bacteriorhodopsin gene were used to clone the archaerhodopsin (aR) gene of Halorubrum xinjiangense strain BD-1^T, and this gene was sequenced and functionally expressed in Escherichia coli. Recombinant E. coli cells harboring the plasmid carrying this gene became slightly purple or blue depending on whether they were supplemented with all- trans retinal or 3,4-dihydroretinal, respectively, during induction with IPTG. The purple and blue membranes from the recombinant E. coli showed maximal absorption at 555 and 588 nm, respectively, which are different from maximal absorption at 568 nm of the wild-type purple membrane. Purple membranes from the recombinant E. coli and from strain BD-1^T were investigated in parallel. The E. coli purple membrane was fabricated into films and photoelectric responses were observed that depended on the light-on and light-off stimuli.     

Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli

Summary The entire structural gene for tyrocidine synthetase 1 from Bacillus brevis ATCC 8185 has been cloned and expressed in Escherichia coli. Transformed E. coli cells were screened for their ability to produce tyrocidine synthetase 1 by in situ immunoassay using antibodies against gramicidin S synthetase 2 which cross-react with tyrocidine synthetase 1. The cloned gene is within a 5.2 kb fragment of B. brevis genomic DNA and requires no external promoter for its expression in E. coli. It was also observed that cloning of the 5.2 kb insert in the opposite orientation still resulted in a high level of tyrocidine synthetase 1 expression in transformed E. coli cells. In addition, protein blotting and partial purification of the gene product by gel filtration revealed a major protein of molecular weight about 100,000 with specific d-phenylalanine dependent ATP-³²PP_i and 2deoxy ATP-³²PP_i exchange activities. These unique activities of tyrocidine synthetase 1 were not detected in protein extracts of E. coli strains carrying the vector.     

Cloning and characterization of the recA gene of Aquaspirillum magnetotacticum

The recA gene of Aquaspirillum magnetotacticum has been isolated from a genomic library and introduced into a recA mutant strain of Escherichia coli K12. The cloned gene complemented both the recombination and DNA repair deficiency of the host and its protein product promoted the proteolytic cleavage of the LexA protein. A protein whose molecular weight is similar to that of the RecA protein of E. coli was associated with the cloned sequence.This paper is affectionately dedicated to Prof. John L. Ingraham     

A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase

The narB gene from the cyanobacterium Synechococcus sp. PCC 7942 was cloned downstream from the LacI-regulated promoter P_trc in the Escherichia coli vector pTrc99A, rendering plasmid pCSLM1. Addition of isopropyl--D-thiogalactoside to E. coli (pCSLM1) resulted in the parallel expression of a 76 kDa polypeptide and a nitrate reductase activity with properties identical to those known for nitrate reductase isolated from Synechococcus cells. As is the case for nitrate reductase from Synechococcus cells, either reduced methyl viologen or reduced ferredoxin could be used as an electron donor for the reduction of nitrate catalyzed by E. coli (pCSLM1) extracts. This data shows that narB is a cyanobacterial structural gene for nitrate reductase.     

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

In vivo cloning ad characterization of mutations of the regulatory locus ompR of Escherichia coli K12

Summary The product of the ompR gene of E. coli K12 is a positive regulatory protein, which is needed for the expression of the major outer membrane proteins OmpC and OmpF in E. coli K12. A simple in vivo technique was used to transfer three ompR mutations (ompR101, ompR472, ompR4) onto a multicopy plasmid carrying the wild-type ompR gene. The resulting clones were transformed into wild type and corresponding mutant back-grounds to analyze their effects on ompC and ompF expression. All of the cloned ompR mutant alleles exhibited a dominant OmpC^- phenotype in an ompR ⁺background. In addition negative complementation of ompF expression was observed between chromosomal ompR4 and multicopy ompR101 alleles. The results suggest an interaction between different OmpR molecules, and thereby support the idea that OmpR can exist as a multimeric protein.     

Partial complementation of the UV sensitivity of E. coli and yeast excision repair mutants by the cloned denV gene of bacteriophage T4

Summary The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors. The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E. coli and in yeast. Induction of the TAC promoter with IPTG resulted in overexpression of the gene, which was lethal to E. coli. Expression of the TACdenV gene in the absence of IPTG, or the use of the yeast GAL1 or ADH promoters resulted in partial complementation of the UV sensitivity of uvrA, uvrB, uvrC and recA mutants of E. coli and rad1, rad2, rad3, rad4 and rad10 mutants of S. cerevisiae. The extent of denV-mediated reactivation of excision-defective mutants was approximately equal to that of photoreactivation of such strains. Excision proficient E. coli cells transformed with a plasmid containing the denV gene were slightly more resistant to ultraviolet (UV) radiation than control cells without the denV gene. On the other hand, excision proficient yeast cells were slightly more sensitive to killing by UV radiation following transformation with a plasmid containing the denV gene. This effect was more pronounced in yeast mutants of the RAD52 epistasis group.     

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation

Summary A DNA fragment that codes for the 364 amino-terminal amino acid residues of a putative Bacillus subtilis SecA homologue has been cloned using the Escherichia coli SecA gene as a probe. The deduced amino acid sequence showed 58% identity to the aminoterminus of the E. coli SecA protein. A DNA fragment which codes for 275 amino-terminal amino acid residues of the B. subtilis SecA homologue was expressed in E. coli and the corresponding gene product was shown to be recognized by anti-E. coli SecA antibodies. This polypeptide, although only about 30% the size of the E. coli SecA protein, also restored growth of E. coli MM52 (secA ^ts) at the non-permissive temperature and the translocation defect of proOmpA in this mutant was relieved to a substantial extent.