Coli DH5alpha Research Articles

Cloning of the PacB-Te r Region from Plasmid Mip233 (IncHI3) and Their Expression in E. coli Ton, Tol Mutants

A region of the plasmid Mip233 (incompatibility group HI3) encoding the phenotypes of resistance to the channel-forming colicins (character PacB) and potassium tellurite (Ter), was cloned and studied. Both properties are contained in an insert of 2.2 Kbp, being the smallest functional clone (pB22) isolated so far. E. coli DH5 alpha pB22 transformants exhibit resistance to the colicins as well as to high levels of tellurite (> 1000 micrograms ml-1). Results suggest that they are genetically linked forming an inducible operon. pB22 does not show significant homology with DNA from other H plasmids. Tests using E. coli ton and tol mutants harbouring recombinant pB22 indicate that the product of gene tolC, but not that of tonB, is required for the expression of the PacB and Ter phenotypes.

Improved electro-transformation of highly DNA-restrictive corynebacteria with DNA extracted from starved Escherichia coli.

Differences of up to 33 000-fold in electro-transformability of highly DNA restrictive corynebacteria are observed in the DNA of a shuttle plasmid extracted from Escherichia coli hosts propagated in different nutritional conditions. Growth of the host in minimal medium increases plasmid transformability, whereas growth on rich media decreases it. In the E. coli DH5 alpha host, the starvation-dependent increase DNA transformability is reverted by supplementing with methionine, an obligate 5-adenosyl-methionine (SAM) precursor. This suggests that an E. coli nutritionally modulated SAM-dependent DNA-methyltransferase may be involved in this phenomenon.

Analysis of Plasmids Cloned from a Virulent Avian Escherichia coli and Transformed into Escherichia coli DH5a

Three of four plasmids from a virulent wild-type avian Escherichia coli were cloned or transformed into an avirulent laboratory recipient E. coli DH5 alpha and tested for the ability to confer a virulence phenotype. The three plasmids transformed into E. coli DH5 alpha were 5, 6, and 56 kb. A fourth plasmid of 64 kb was not successfully transformed. Parameters used to measure virulence included presence of type 1 pili and a smooth lipopolysaccharide (LPS) layer, motility, production of Colicin V, resistance to host complement, and embryo lethality. The 5-kb plasmid encoded for ampicillin resistance, whereas the 6-kb plasmid encoded for tetracycline resistance. The 56-kb plasmid encoded for streptomycin, sulfisoxazole, and tetracycline resistance. Twelve-day embryos inoculated with 467 colony-forming units of E. coli DH5 alpha containing the 56-kb plasmid had increased death rates (45%) in the embryo lethality assay and a decreased weight of surviving embryos with cranial hemorrhages as compared with embryos inoculated with similar amounts of E. coli DH5 alpha (0%) and phosphate-buffered saline (0%). Embryos inoculated with the wild-type virulent E. coli had 90% deaths. The 56-kb plasmid also had homology with a probe for Colicin V production (cvaC). No differences in LPS layer, complement resistance, motility, Colicin V activity, type 1 pili, cell-free supernatant proteins, or outer membrane proteins were observed in the transformants when compared with nontransformed E. coli DH5 alpha.

Cloning and expression of rfb genes from Vibrio anguillarum serotype O2 in Escherichia coli: evidence for cross-reactive epitopes

Vibrio ordalii and Vibrio anguillarum O2 express lipopolysaccharide (LPS) O antigens containing both specific and cross-reactive epitopes. The localization of these epitopes on the O antigen is not known. We have cloned and expressed the rfb gene cluster for O-antigen synthesis from V. anguillarum O2 (rfbVaO2) in Escherichia coli. E. coli DH5 alpha containing the recombinant plasmid pAM86 expressed O antigens which reacted with polyclonal antisera to V. ordalii and to V. anguillarum O2 LPS and with monoclonal antibody (MAb) 7B4, which is specific for V. anguillarum O2 O antigens. The recombinant strains were also protected from bactericidal killing by normal fish serum. Surprisingly, the LPS expressed from the cloned rfbVaO2 genes also reacted with MAb A16, which is specific for V. ordalii O antigens. Western immunoblot analysis revealed that MAb 7B4 reacted with recombinant LPS bearing shorter O-antigen repeat units, while MAb A16 reacted with the longer O antigens. Similar results were obtained when pAM86 was transformed into E. coli CLM4, which has a deletion spanning the sbcB-rfb region, indicating that the changes in antigenic profiles of O antigens from the recombinant strains were not due to genes within the E. coli rfb cluster. These data suggest that the epitope recognized by the MAb A16 is expressed by V. anguillarum O2 strains but it is apparently not accessible to the antibody in the native O polysaccharide. Cloning of the rfbVaO2 gene cluster resulted in expression of a novel O antigen. The modification(s) which leads to the alterations in antigenic profile of these recombinant LPS remains to be determined.

Discovery of a ptsHl operon, which includes a third gene (ptsT), in the thermophile Bacillus stearothermophilus

The discovery of ptsHI operon in Bacillus stearothermophilus XL-65-6 coupled with our previous report of a cel operon (Lai & Ingram, J Bacteriol 175, 6441-6450, 1993) demonstrates that this thermophilic organism contains all of the genes required for cellobiose uptake by the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Genes encoding the two general PTS proteins, HPr (ptsH) and enzyme I (ptsI), were cloned and sequenced. These form an operon which includes a third small gene (ptsT) of unknown function (encoded product M(r) 18428). Both ptsH and ptsI were expressed at high levels from a single plasmid in Escherichia coli and complemented corresponding host mutations. Although the translated sequences for these genes were similar to homologues from Gram-positive mesophiles (64-77% identity), the B. stearothermophilus gene products were unusual in having a higher predicted pI and fewer negatively charged amino acid residues. Enzyme I also contained more alanine and leucine than mesophilic counterparts. Interestingly, ptsT inhibited the growth of E. coli ptsI mutants at 37 degrees C. No such inhibition was observed during incubation at a lower temperature (30 degrees C) or in E. coli DH5 alpha, which is wild-type for ptsI. The predicted translation product from ptsT contained a high proportion of basic amino acids (27%) and had a high predicted pI (pH 11.7), properties similar to bacterial histone-like proteins, but did not exhibit homology to any sequences in the current database. Regions upstream and downstream from the ptsHI operon contain genes with homology to Bacillus subtilis ptsG and wapA (wall-associated protein), respectively.

Corynebacterium glutamicumDNA is subjected to methylation-restriction inEscherichia coli

Efficient electroporation of Escherichia coli with plasmid DNA isolated from Corynebacterium glutamicum depends on the use of Mcr-deficient E. coli strains. The transformation frequency increased nearly 800-fold when the Mcr-deficient E. coli DH5 alpha MCR was used instead of E. coli DH5 alpha. We used E. coli strains with different mutations in the methyl-specific restriction systems to show that McrBC-deficiency is sufficient to generate this effect. The results imply that C. glutamicum DNA contains methylcytosine in specific sequences recognized by the E. coli McrBC system.

Epithelial cell invasion by bovine septicemic Escherichia coli.

Little is known regarding the pathogenesis of Escherichia coli-induced septicemic colibacillosis of calves. To understand the mechanism by which these strains penetrate the intestinal epithelium and gain access to the bloodstream, we examined the potential of bovine septicemic E. coli to invade cultured epithelial cells. By using a gentamicin survival assay, we demonstrated bacterial invasion of Madin-Darby canine kidney (MDCK) cells. Transcytosis of polarized MDCK cell monolayers was also observed, but only when bacteria were added to the basolateral surface. Electron microscopy confirmed the presence of intracellular organisms which appeared to be within membrane-bound vacuoles. The bovine septicemic isolate used in this study expressed the fimbrial adhesion CS31A. To examine the role of CS31A-mediated adherence in invasion and transcytosis of MDCK cell monolayers, a CS31A-deficient mutant was constructed by suicide vector-mediated insertional mutagenesis. Although nonadherent, the mutant showed a level of invasion similar to that of the wild-type parent. E. coli DH5 alpha carrying the cloned CS31A determinant was noninvasive. These findings suggest that expression of CS31A is neither required nor sufficient to mediate invasion.

Construction and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative form of viruslike particle CAK1 isolated from Clostridium acetobutylicum NCIB 6444

A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constructed by ligating 5-kbp Escherichia coli plasmid pAK102 (AprEmr) and the 6.6-kbp HaeIII-linearized replicative form of the CAK1 viruslike particle from Clostridium acetobutylicum NCIB 6444. Phagemid pCAK1 (11.6 kbp) replicated via the ColE1 replication origin derived from pAK102 in E. coli. Single-stranded DNA (ssDNA) molecules complexed with protein in a manner which protected ssDNA from nucleases were recovered from the supernatant of E. coli DH11S transformants containing pCAK1 in the absence of cell lysis. This suggests that the viral-strand DNA synthesis replication origin of CAK1 and associated gene expression are functional in E. coli DH11S. The single-stranded form of pCAK1 isolated from E. coli supernatant was transformed into E. coli DH5 alpha' or DH11S by electroporation. Isolation of ampicillin-resistant E. coli transformants following transformation suggests that the complementary-strand DNA synthesis replication origin of CAK1 is also functional in E. coli. The coat proteins associated with ssDNA of pCAK1 demonstrated sensitivity to proteinase K and various solvents (i.e., phenol and chloroform), similar to the results obtained previously with CAK1. Following phagemid construction in E. coli, pCAK1 was transformed into C. acetobutylicum ATCC 824 and C. perfringens 13 by intact cell electroporation. Restriction enzyme analysis of pCAK1 isolated from erythromycin-resistant transformants of both C. acetobutylicum and C. perfringens suggested that it was identical to that present in E. coli transformants.

High-affinity iron uptake systems present in Erwinia carotovora subsp. carotovora include the hydroxamate siderophore aerobactin.

The phytopathogenic bacterium Erwinia carotovora subsp. carotovora W3C105 produced the hydroxamate siderophore aerobactin under iron-limiting conditions. A survey of 22 diverse strains of E. carotovora revealed that strain W3C105 alone produced aerobactin. The ferric-aerobactin receptor of strain W3C105 was an 80-kDa protein, identified by immunoblots of Sarkosyl-soluble proteins obtained from E. carotovora cells grown in iron-depleted medium and probed with antiserum raised against the 74-kDa ferric-aerobactin receptor encoded by the pColV-K30 plasmid of Escherichia coli. Genes determining aerobactin biosynthesis and uptake were localized to an 11.3-kb EcoRI-HindIII chromosomal fragment of strain W3C105. A 10-kb subclone of the fragment conferred on E. coli DH5 alpha both aerobactin biosynthesis and uptake, determined by cloacin DF13 sensitivity, the presence of the 80-kDa receptor protein, and iron-independent growth of E. coli clones. The aerobactin biosynthesis genes of E. carotovora W3C105 hybridized to those of the pColV-K30 plasmid of E. coli, but the restriction patterns of the aerobactin regions of E. coli and E. carotovora differed. Although the aerobactin region of enteric bacteria is commonly flanked by IS1-like sequences, IS1 sequences were not detected in the genomic DNA or the cloned aerobactin region of E. carotovora. E. coli DH5 alpha cells harboring cloned aerobactin biosynthesis genes from E. carotovora W3C105 produced greater quantities of aerobactin and the 80-kDa ferric-aerobactin receptor when grown in iron-limited than in iron-replete medium. Strain W3C105 grew on an iron-limited medium, whereas derivatives that lacked a functional aerobactin iron acquisition system did not grow on the medium. These results provide evidence for the occurrence and heterogeneity of aerobactin as a high-affinity iron uptake system of both clinical and phytopathogenic species of the Enterobacteriaceae. Although future studies may reveal a role for aerobactin in the virulence or ecology of strain W3C105, a functional aerobactin iron acquisition system is not necessary for the pathogenicity of E. carotovora.

Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonasmarginalis N6301

A pectin lyase (PNL;EC4.2.2.10) gene of Pseudomonas marginalis N6301 was cloned and expressed in Escherichia coli. We purified PNL from P. marginalis N6301 and determined N-terminal 33 amino acids sequence. From this sequence, we synthesized two oligonucleotide probes. From the analysis of Southern hybridization, 2. 1kb EcoRI-SmaI fragment from the chromosomal DNA of P. marginalis was found to hybridize with oligonucleotide probes. Then, we cloned the fragment into pUC119 vector and transformed into E. coli DH5 alpha. A plasmid thus obtained was designated as pPNL6301. E. coli DH5 alpha harboring pPNL6301 expressed PNL activity. The nucleotide sequence of pn1 gene in the plasmid pPNL6301 encoding PNL from P. marginalis N6301 was determined. The structural gene of pn1 consisted of 936 base pairs. An open reading frame that encodes a 34,103 dalton polypeptide composed of 312 amino acids was assigned. The molecular weight of the polypeptide predicted from the amino acid composition was close to that of PNL of P. marginalis N6301 determined. The nucleotide sequence of the 5'-flanking region of pn1 gene showed the presence of the consensus sequence of LexA binding site, Pribnow box and ribosome binding site as found in Escherichia coli. The amino acid sequence homology of PNLs and nucleotide sequence homology of pn1 gene between P. marginalis N6301 and E. carotovora Er were 60.8% and 57.2%, respectively.

Cloning and expression in Escherichia coli of a gene encoding superoxide dismutase from Listeria ivanovii.

A chromosomal DNA fragment from the gram-positive bacterium Listeria ivanovii (ATCC 19119) encoding a superoxide dismutase (SOD) gene has been cloned in Escherichia coli QC779 (sodAsodB) using the plasmid vector pTZ19R. The DNA fragment inserted into the plasmid showed high structural instability in E. coli QC779 (recA+), but turned out to be a stable 1.95 kbp DNA fragment when transformed into E. coli DH5 alpha (recA-). The gene is expressed in both of these E. coli strains at high levels. Preliminary studies showed that the activity of the recombinant SOD within E. coli DH5 alpha was up to 13-times the combined activity of both E. coli SODs. The recombinant SOD forms active hybrid SODs with both E. coli SODs in vivo.